Substance P and somatostatin neurons limit facial itch by recruiting distinct nociceptive circuits in the brainstem

Joshua J Wheeler; Nidha Williams; Aditi Vigneshwar; B Duncan X Lascelles; Thierry Olivry; Santosh K Mishra

doi:10.1038/s42003-026-10128-9

. 2026 May 2;9:916. doi: 10.1038/s42003-026-10128-9

Substance P and somatostatin neurons limit facial itch by recruiting distinct nociceptive circuits in the brainstem

Joshua J Wheeler ^1,², Nidha Williams ¹, Aditi Vigneshwar ^1,³, B Duncan X Lascelles ^3,⁴, Thierry Olivry ¹, Santosh K Mishra ^1,^2,^4,^5,^✉

PMCID: PMC13341877 PMID: 42069949

Abstract

Itch is a complex noxious sensation associated with many skin and systemic conditions, which varies in intensity and quality across different body regions. Despite its prevalence, the molecular and cellular mechanisms underlying regional itch differences remain poorly understood. Investigating the neural basis of regional itch differences, we identified a functional divergence in neuropeptide signaling and central circuit engagement between the trigeminal and spinal systems, which was independent of peripheral innervation density. Utilizing a combination of behavioral, pharmacological, genetic, and molecular assays, we identified a unique population of trigeminal (TG) neurons that facilitate specialized itch-pain coding. Our results indicate that while histamine receptors HRH1 and HRH3 are both involved in mediating mixed itch-and-pain sensations, the specific activity of Substance P (SP)- and Somatostatin (SST)-expressing neurons orchestrates this transition in the cheek. This behavioral shift is mediated by a central mechanism wherein sensory neurons activation recruits distinct nociceptive circuits within the brainstem. In brief, these findings provide insights into the molecular and cellular mechanisms underlying regional itch differences, highlighting the importance of considering anatomical location when developing targeted treatments.

Subject terms: Molecular neuroscience, Somatic system

Trigeminal itch is driven by a unique population of sensory neurons where Substance P and Somatostatin signaling recruit specialized brainstem circuits to transform histamine-evoked sensations into a distinct mix of itch and pain.

Introduction

Somatosensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) detect various stimuli, generating distinct sensations and responses. DRG neurons innervate the skin of the limbs and torso, whereas TG neurons innervate unique sensory areas in the head and neck, including the eyes, nose, mouth, meninges, and vibrissae. Reports have identified genes linked to somatosensory neurons’ detection specificity in DRG and TG. DRG RNA-sequencing reveals distinct expression profiles for proprioceptive and itch-responsive neurons predominantly in mice^1,2 and humans^3–5. However, TG neuronal populations differ from DRG, as they lack proprioceptive neurons and innervate several unique sensory environments: the eyes, nasal passages and sinuses, meninges, and, in animals, vibrissae. Recent research has found TG-specific differences at the transcription level^4,6,7, but whether that drives a differential behavioral response or differences in itch circuitry in mice is unknown.

In humans, the severity of itch is perceived differently across body regions, with the face and torso^8–10 exhibiting distinct intensity thresholds. Animal models have recapitulated these phenomena, showing fewer scratching bouts in response to intradermal pruritogen injections in the cheek than dorsal nape^11,12. This difference is consistent across common pruritogens^11–19, including histamine^11,12. Notably, facial injection of pruritogens and algogens activates a partially overlapping population of interneurons found in the spinal trigeminal nucleus¹⁷. In contrast, DRG itch and pain signals primarily activate separate populations of interneurons^3,19,20. The distinct neural coding in the TG versus the DRG suggests the involvement of specific neuropeptides and signaling pathways involved in transmitting itch signals from the face versus the body. While itch sensations are critically propagated by brain natriuretic peptide (BNP)/somatostatin-expressing neurons (NP3), they are also transduced by distinct populations of MrgprD-expressing (NP1) and MrgprA3-expressing (NP2) pruriceptors, which respond to beta3-alanine and chloroquine, respectively^20,21. Activation of these sensory neurons triggers the release of neuropeptides such as SP and BNP. In the spinal dorsal horn, these neuropeptides activate specific interneuron circuits to process these pain and itch sensations^19–22. However, whether the trigeminal system utilizes these same labeled lines or employs a more integrated signaling strategy to process facial sensations remains an area of active investigation.

Here, we investigated the mechanisms underlying regional differences in pruriceptive processing between the face and the body using a multifaceted approach combining mouse genetics with behavioral, pharmacological, and molecular techniques. We first identify the behavioral divergence between the dorsal nape and cheek in response to histaminergic and non-histaminergic pruritogens. Next, we utilized local pharmacological antagonism and mast cell ablation to isolate the role of peripheral sensory neurons, alongside experiments in mice lacking B-type natriuretic peptide (BNP) or Substance P (SP). These findings are corroborated by transcriptional activation data showing distinct recruitment in the TG compared to the DRG. Furthermore, we investigated the central circuits involved by mapping the activation of TacR1-expressing and NPR1-expressing interneurons within the trigeminal nucleus caudalis (TNC or Sp5C) via immediate early genes (Egr1 or cFos) expression. By comparing molecular profiles in human tissues, we validated the translational relevance and conservation of these mechanisms in the human sensory system. Collectively, these studies identify a mandatory synergy between histamine receptor subtypes and neuropeptidergic signaling that distinguishes facial itch from that of the body.

Results

Regional scratching responses differ between the face and neck

We initially performed a literature review (Supplemental Data) of papers published between January 2009 and June 2023 (Fig. 1B, C) which revealed significant differences in scratching bouts when normalized for time between these two regions in response to various pruritogens, including for histamine (*p < 0.0001), chloroquine (*p = 0.0008), and SLIGRL-NH2, a PAR-2 specific agonist (*p = 0.0006) (Fig. 1B). These data were further confirmed when we combined all scratching bouts/minute values for all pruritogens (*p = 0.0073) (Fig. 1C). Based on this systematic review, we sought to compare the behavior responses of these two regions to confirm these results using two common pruritogens: histamine¹¹ and chloroquine¹⁴.

To investigate the regional scratching response between the face and neck, we comprehensively analyzed scratching behavior in C57Bl6/J mice following intradermal injections of pruritogens into the dorsal nape and cheek. Histamine elicited significantly fewer scratch bouts of ipsilateral cheek-directed scratching than dorsal nape-directed scratching bouts (*p = 0.0040) (Fig. 1D) in C57Bl6/J mice injected with histamine. In contrast, we did not find any significant differences when comparing the total amount of time spent scratching directed at these areas (*p = 0.2947) (Fig. 1E), and no significant differences were seen in the average bout duration (*p = 0.2540) (Fig. 1F). The time course of histamine-induced scratching bouts differed significantly between the dorsal nape and cheek (Fig. 1G). Ipsilateral cheek-directed scratching bouts peaked within the first 5 min (*p < 0.0001) (Fig. 1G) and the number of dorsal nape-directed scratching bouts peaked at 15 min (*p = 0.0001) (Fig. 1G). We found similar results using chloroquine (CQ) in C57Bl6/J mice. In C57Bl6/J mice, CQ elicited significantly fewer scratching bouts in the ipsilateral cheek as compared to the dorsal nape (*p = 0.0332) (Fig. 1L) and significantly reduced total scratching times (*p = 0.0164) (Fig. 1M). As with histamine, the average scratch bout duration did not change between the two injection regions following i.d. CQ (Fig. 1N). Unlike histamine, CQ only elicited significantly more scratching bouts in the dorsal nape 15 min after injection (*p = 0.0025) (Fig. 1O).

To confirm that our findings in C57Bl6/J mice were not strain-specific, we repeated the experiments using CD-1 mice, a commonly used itch research outbred mouse line (Fig. 1H–K)²³. Our histamine (i.d) results showed a similar pattern: fewer ipsilateral cheek-directed scratching bouts (*p < 0.0001); however, we found significantly shorter scratching times than dorsal nape-directed scratching bouts (*p < 0.0001) (Fig. 1H, I). Unlike with C57BL/6J mice, bout duration significantly differed between regions in the CD-1 mice (*p = 0.0380) (Fig. 1J). The time course of histamine-induced scratching bouts differed between regions, with cheek-directed scratching peaking within 5 min, and significant differences were observed at 15 (*p = 0.0002), 20 (*p = 0.0477), and 30 min (*p = 0.0009) (Fig. 1K). We also injected CQ (i.d.) into the dorsal nape and cheek of CD-1 mice to again confirm that these results were not strain-specific. As expected, CD-1 mice performed significantly fewer scratching bouts directed at the ipsilateral cheek as compared to the dorsal nape following CQ injection (*p = 0.0013) (Fig. 1P), which then resulted in these mice spending significantly less time scratching the ipsilateral cheek overall (*p = 0.0007) (Fig. 1Q). Unlike histamine, CQ injection did not cause the average bout duration to change between the two injection regions in CD-1 mice (Fig. 1R). Finally, CQ injection into the nape caused significantly more bouts of scratching immediately following injection and continuing for 15 min (*p < 0.0001 [0–5 min], *p = 0.0043[5–10 min], and *p = 0.0014 [10–15 min]) (Fig. 1S). Overall, our data suggests that regional differences in itch are conserved across outbred and inbred mouse lines, with the cheek having a significantly reduced number of histamine- and chloroquine-induced scratching bouts compared to the dorsal nape, indicating that the face is less responsive to histamine- and CQ-induced itch.

Regional itch variations are not explained by peripheral innervation densities

Given the difference in histamine-induced itch between the dorsal nape and cheek, we hypothesized that there may be a difference in innervation density of itch-sensing neurons between the facial dermatomes innervated by the TG²⁴ and the dorsal nape dermatomes innervated by the DRG²⁵. To assess this, we generated SST-cre::Ai9 mice in which tdTomato was conditionally expressed in SST-expressing neurons. We chose to generate SST-cre::Ai9 mice since previous work has demonstrated that SST-expressing DRG sensory neurons are one sensory neuron population, the other being MrgprA3¹⁸, responsible for mediating itch signals induced by histamine^20,26. We found that the skin of the cheek has a significantly higher degree of innervation by SST-cre::Ai9 neurons as compared to the dorsal nape (*p = 0.0158) (Fig. 2A–C). As an additional control, we also stained these sections for PGP9.5, a pan-neuronal marker for cutaneous innervation²⁷ and found a significant increase in overall innervation in the cheek as compared to the dorsal nape (*p = 0.0132) (Fig. 2A–D). Due to the increased innervation density of SST-cre::Ai9 afferents in the cheek, we hypothesized that there was an increase in the number of SST-expressing neurons in the TG. As expected, we found significantly more SST-expressing neurons in the TG as compared to the DRG (*p = 0.0029) (Fig. 2E–G). This increase is likely to be responsible for increased SST-positive afferent density between the dorsal nape and the cheek. Our findings demonstrate that the observed reduction in scratching in the cheek is not correlated with differences in overall innervation density or the density of itch-sensing neurons.

Fig. 2 — A Representative confocal images of skin isolated from the dorsal nape of naïve SST-cre::Ai9 mice. B Representative confocal images of skin isolated from the cheek of naïve SST-cre::Ai9 mice. C Quantification of SST intraepidermal nerve fiber innervation density of the dorsal nape and cheek of SST-cre::Ai9 mice. D Quantification of PGP9.5 intraepidermal nerve fiber innervation density of the dorsal nape and cheek of SST-cre::Ai9 mice. E Representative image of SST expression in the DRG. F Representative image of SST expression in the TG. G Quantification of SST-positive neurons in the DRG and TG, normalized to Tuj staining. Data is presented as Mean ± Standard Deviation. Each dot represents one biological replicate. *p < 0.05 as determined by a paired 2-tailed Student’s t-test. Scale bar = 100 µm. A minimum of 300 cells/mouse/ganglia was counted.

Distinct role of SP in modulating itch response in the cheek and dorsal nape

Since innervation density did not explain the regional differences in itch response, we hypothesized that pain-associated neuropeptides, specifically Substance P (SP)²⁸, might modulate ipsilateral cheek-directed scratching behavior. Based on previous observations, we found that in the dorsal nape, all SST-expressing DRG sensory neurons are positive for BNP²⁰. Further, these BNP-expressing DRG sensory neurons are not positive for Substance P (SP)²¹, a canonical pain-associated neuropeptide²⁸. While the co-expression of Substance P (SP) and Brain Natriuretic Peptide (BNP) has been shown to be mutually exclusive in the DRG²¹, their co-expression status in the TG remains uncharacterized. To test if SP modulated itch responses in the cheek, we used knockout (KO) mice lacking SP or BNP and quantified scratching bouts after histamine injection into both the dorsal nape and the cheek (Fig. 3). We found that SP-KO mice exhibited significantly more ipsilateral cheek-directed scratching bouts than wild-type controls (*p = 0.0007) (Fig. 3A), with no differences in dorsal nape-directed scratching bouts (*p = 0.2390) (Fig. 3B). We used BNP-KO mice as a control, as these mice should lack a scratching behavior phenotype, and demonstrated that the loss of BNP resulted in a significant decrease in the total number of dorsal nape (*p = 0.0126) and ipsilateral cheek-directed (*p = 0.0338) scratching bouts (Fig. 3E, F). To confirm our histamine-induced scratching behavior results in SP-KO and BNP-KO mice, we pharmacologically inhibited the SP receptor (TacR1) with L-733,060^29,30. We found that these mice exhibited significantly higher amounts of ipsilateral cheek-directed scratching bouts than vehicle-treated mice (*p = 0.0198) (Fig. 3C). Like the SP-KO mice, L-733,060 treated mice had no significant differences in dorsal nape-directed scratching bout compared to vehicle-treated mice (*p = 0.3568) (Fig. 3D). Mice treated with the NPR1 antagonist JS-11³¹ experienced significantly fewer scratching bouts in both the ipsilateral cheek (*p = 0.0338) and the dorsal nape (*p = 0.0396) (Fig. 3G, H). These findings suggest that SP plays distinct roles in modulating itch response in the cheek and dorsal nape, with SP inhibiting and BNP promoting itch in the cheek.

Fig. 3 — A Injection region schematic. B Substance P KO mice had significantly more ipsilateral cheek-directed scratching bouts as compared to WT controls following intradermal histamine (100 µg/20 µL) injection. C There were no significant differences in the total number of dorsal nape-directed scratching bouts between SP-KO mice and WT controls following intradermal histamine (100 µg/20 µL) injection. D Pharmacological inhibition of TacR1 with L-733,060 (1 mg/kg in 100 µL I.V.) significantly increased the total number of ipsilateral cheek-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection but did not affect histamine-induced scratching behavior when injected intradermally into the ipsilateral cheek skin. E Pharmacological inhibition of TacR1 with L-733,060 (1 mg/kg in 100 µL I.V. or 1 mg/kg in 50 µL i.d.) did not significantly change the number of dorsal nape-directed scratching bouts following intradermal histamine (100 µg/20 µL). F BNP-KO mice exhibited significantly fewer ipsilateral cheek-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection. G BNP-KO mice exhibited significantly fewer dorsal nape-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection. H Intraperitoneal administration of JS-11 (163 µg in 50 µL) did not significantly reduce the total number of ipsilateral cheek-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection. I Intraperitoneal administration of JS-11 (163 µg in 50 µL) significantly reduced the total number of dorsal nape-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection. J No significant differences in scratching behavior were seen in the cheek of mast cell-deficient mice (Cre + /DTA + ) as compared to genetic control mice following intradermal histamine (100 µg/20 uL) injection. K Mast cell-deficient mice did not have any significant differences in scratching behavior as compared to genetic control mice following intradermal histamine (100 µg/20 µL) injection. Data is presented as Mean ± Standard Deviation. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by a 2-tailed Student’s t-test. Each data point represents 1 biological replicate. Cartoon in (A) was generated using BioRender; https://BioRender.com/7jv1vlp.

While our global knockout data suggested that SP is required for this itch-pain response, we sought to determine whether this effect was mediated by peripheral skin cells or central neural circuits. To rule out the role of the skin in transducing this itch, we performed two targeted experiments focusing on local receptors and mast cells. First, we locally co-inject L-733,060 with histamine into the two regions of interest (Fig. 3D, E). We did not find any significant changes in scratching behavior with local co-injection of L-733,060. In a second set of experiments, we generated MCPT5-cre::ROSA^DTA mice (Cre+/DTA+) to ablate cutaneous mast cells. Mice with ablated mast cells did not exhibit any significant differences in scratching behavior in the cheek or nape (Fig. 3J, K) when compared to littermate control mice (Cre-/DTA+). This provides strong evidence for cellular and spatial specificity, indicating that the requirement for SP in this model is not mediated by peripheral itch-sensitive cells but rather by sensory release of neuropeptide to the downstream in the CNS for generating sensation.

Histamine receptor-specific itch induction

To investigate which histamine receptors mediate histamine-induced itch in these regions, we built on our previous findings on regional differences in itch between the cheek and nape. We focused on the four histamine receptors, three of which are expressed in DRG sensory neurons. scRNA-sequencing data showed that HRH1 mRNA is expressed predominantly in SST- and MrgprA3-positive DRG sensory neurons involved in itch, while HRH3 is expressed in SP-positive DRG sensory neurons involved in pain². To determine which histamine receptor(s) are responsible for transducing histamine-induced itch, we injected 2-pyridylethylamine (HRH1-specific agonist)³² and immethridine (HRH3-specific agonist)³³ into the dorsal nape and cheek of mice. We found that 2-pyridylethylamine induced HRH1-mediated itch in both the dorsal nape and ipsilateral cheek, with no significant differences in scratching bouts compared to histamine-injected mice (*p = 0.5208, nape; *p = 0.7708, cheek) (Fig. 4B, C). We found that while both 2-Pyr and histamine induced cheek-directed scratching, 2-Pyr elicited significantly more pain-related cheek wipes than histamine (*p = 0.0230) (Fig. 4D). Immethridine also induced more ipsilateral cheek wipes (*p = 0.136) (Fig. 4D), but fewer scratching bouts than histamine (*p < 0.0001, nape; *p = 0.0264, cheek) in both regions. Immethridine induced fewer scratching bouts than 2-pyridylethylamine in the nape (*p = 0.0002, nape), but not the cheek (*p = 0.2373). Lastly, to determine which nocifensive response is mediated by which histamine receptor, we used the HRH1 antagonist, diphenhydramine, and the HRH3 antagonist, JNJ-5207852, to specifically block these receptors in mice that then had histamine injected into the cheek. We found that blocking either HRH1 or HRH3 abrogated both itch (scratching) (*p = 0.0031 [diphenhydramine], *p = 0.0003 [JNJ-5207852]) and pain (wiping) (*p = 0.0001 [diphenhydramine], *p = 0.0003 [JNJ-5207852]) responses (Fig. 4C, D). While H1R and H3R have been traditionally associated with itch and pain, respectively, our data suggests a mandatory synergistic role for both receptors in mediating these behaviors.

Fig. 4 — A Schematic demonstrating which histamine receptors are activated by the agonists Histamine, 2-pyridylethylamine, or immethridine. B immethridine (100 µg/20 µL) induces significantly fewer scratching bouts than histamine (100 µg/20 µL) or 2-Pyridylethylamine in the dorsal nape. C immethridine (100 µg/20 µL) induces significantly fewer scratching bouts than histamine (100 µg/20 µL) or 2-Pyridylethylamine in the ipsilateral cheek. D immethridine (100 µg/20 µL) induces significantly more ipsilateral cheek-directed wiping bouts than histamine (100 µg/20 µL). Antagonism of either HRH1 by diphenhydramine or HRH3 by JNJ-5207852 blocked both itch and pain responses (C and D). Data is presented as Mean ± Standard Deviation, *p < 0.05, ***p < 0.001 as determined by a 1-way ANOVA with a *posthoc* Holm – Šídák test for multiple comparisons. The schematic in (A) was made in BioRender; https://BioRender.com/uje57y7.

Regional variations in sensory neuron populations underlying itch and pain

Our previous findings on regional differences in itch and SST/SP innervation led us to investigate the populations of SST-, SP-, MrgprA3-, HRH1-, and HRH3-expressing sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) of mice, which we also further validated in humans. Our SST-cre::Ai9 innervation results indicated that there should be a larger proportion of SST-positive TG sensory neurons. Additionally, our behavioral results indirectly indicate that SP-positive TG sensory neurons are important in modulating ipsilateral cheek itch. Since pain signaling is capable of modulating itch sensation and vice versa^19,20, we hypothesized that there is a change in the number of TG sensory neurons that are positive for both SST and SP. We found that overall, there was a significant increase in the percentage of TG sensory neurons positive for both SST and SP as compared to the DRG (*p = 0.0012) (Fig. 5A–E). Since itch is predominantly perceived to occur on the skin, we used the neuronal tracer wheat germ agglutinin (WGA-350) to determine if this increase in TG neurons co-expressing SST and SP occurred in skin-innervating TG neurons. We found a larger population of cheek skin-innervating TG sensory neurons positive for both SST and SP compared to dorsal nape-innervating DRG sensory neurons (*p = 0.0051) (Fig. 5F–H), indicating that this population is important in modulating itch transmission in the TG.

Fig. 5 — A Representative immunohistochemical images of SST and SP-positive DRG neurons isolated from *Tac1*-cre::Ai9 mice that were stained for SST. B Representative immunohistochemical images of SST and SP-positive TG neurons isolated from *Tac1*-cre::Ai9 mice that were stained for SST. C Quantification of neurons positive for both SST & SP, normalized to the total number of SST-positive neurons. D Pie chart showing the percentage of DRG neurons positive for SST-only and both SST and SP. E Pie chart showing the percentage of TG neurons positive for SST-only, and both SST and SP. F Representative ISH images of SST and SP-positive DRG neurons in mice that received intradermal WGA-350 into the dorsal nape. G Representative ISH images of SST and SP-positive TG neurons in mice that received intradermal WGA-350 into the cheek. H Quantification of the SST+/SP+ neuron population in the TG and DRG in mice injected with WGA-350, normalized to the number of SST-positive WGA-350-positive neurons. Data is shown as Mean ± Standard Deviation, **p < 0.01 as determined by a 2-tailed paired Student’s t-test. Arrows identify neurons positive for both SST and SP. Scale bar = 100 µm for all images. Similar results were seen in N = 5 mice. A minimum of 500 cells per mouse per ganglion was counted.

Since we found an increase in skin-innervating, SST-positive neurons that express SP, we next investigated the expression of HRH1, HRH3, and MrgprA3 in skin-innervating sensory neurons in mice (Fig. 6). Of the skin-innervating DRG neurons, only 3% co-expressed HRH1 with SP (Fig. 6A, G). In the TG, the percentage of skin-innervating neurons co-expressing HRH1 and SP increased to 5.2% (Fig. 6B, G). We found a similar trend with skin-innervating neurons co-expressing HRH3 and SP: 8.5% of skin-innervating DRG neurons co-expressed HRH3 and SP, while in skin-innervating TG neurons, this percentage increased to 22.6% (Fig. 6C, D, H). Finally, a similar trend was found for MrgprA3: 2.3% of skin-innervating DRG neurons co-expressed MrgprA3 and SP, while this percentage increased to 16.9% of skin-innervating TG neurons (Fig. 6E, F, I). Also, we found that roughly half of skin-innervating DRG neurons expressing MrgprA3 also expressed SST, and that nearly all skin-innervating TG neurons expressing MrgprA3 also expressed SST (Fig. 6I). To assess the translational relevance of our findings, we performed ISH in human DRG and TG for HRH1, HRH3, SST, and SP. First, we determined that there is an increase in the percentage of TG neurons co-expressing SST and SP (Fig. 7A–E), like what is seen in mice. We also stained human DRG and TG for HRH1 and HRH3 in addition to SST and SP, and found that these populations also underwent similar changes in co-expression in the TG as compared to the DRG (Fig. 7F–M).

Fig. 6 — A Representative images of DRG neurons isolated from mice receiving intradermal WGA-350 (50 µL of 1 mg/mL stock) that were ISH-stained for HRH1, SP, and SST. B Representative images of TG neurons isolated from mice receiving intradermal WGA-350 (50 µL of 1 mg/mL stock) that were ISH-stained for HRH1, SP, and SST. C Representative images of DRG neurons from mice receiving intradermal WGA-350 (50 µL of 1 mg/mL stock) that were ISH-stained for HRH3, SP, and SST. D Representative images of TG neurons from mice receiving intradermal WGA-350 (50 µL of 1 mg/mL stock) that were ISH-stained for HRH3, SP, and SST. E Representative images of DRG neurons from mice receiving intradermal WGA-350 (50 µL of 1 mg/mL stock) that were ISH-stained for MrgprA3, SP, and SST. F Representative images of TG neurons from mice receiving intradermal WGA-350 (50 µL of 1 mg/mL stock) that were ISH-stained for MrgprA3, SP, and SST. G Pie chart showing the sizes of the HRH1, SP, and SST populations normalized to WGA-350-positive DRG or TG neurons. H Pie chart showing the sizes of the HRH3, SP, and SST populations normalized to WGA-350-positive DRG neurons and TG neurons. I Pie chart showing the sizes of the MrgprA3, SP, and SST populations normalized to WGA-350-positive DRG and TG neurons. Scale bar = 100 µm. Percentages shown are the average of 5 male mice. Similar ISH staining patterns were seen in all 5 C57Bl6/J mice. Arrows identify neurons positive for all three genes of interest plus WGA-350. A minimum of 250 cells per mouse per ganglion was counted.

Fig. 7 — A Representative ISH images of human DRG stained for SP and SST. B Representative ISH images of human TG stained for SP and SST. C Quantification of SP + /SST+ neurons normalized to the number of SST+ neurons in each ganglion. D Pie chart showing the percentage of SP+/SST+ neurons and SST+ neurons in human DRG. E Pie chart showing the percentage of SP + /SST+ neurons and SST+ neurons in human TG. F Representative FISH images of Human DRG neurons stained for HRH1, SP, and SST. G Representative FISH images of Human TG neurons stained for HRH1, SP, and SST. H Representative FISH images of Human DRG neurons stained for HRH3, SP, and SST. I Representative FISH images of Human DRG neurons stained for HRH3, SP, and SST. J Pie charts showing the percentages of HRH1, SP, and SST populations normalized to the number of SST + DRG neurons. K Pie Charts showing the percentages of HRH1, SP, and SST populations normalized to the number of SST + TG neurons. L Pie chart showing the percentages of HRH3, SP, and SST populations normalized to the number of SST + DRG neurons. M Pie charts showing the percentages of HRH3, SP, and SST populations normalized to the number of SST + TG neurons. Scale bar = 100 µm. Percentages shown are the average of 2 male human DRG or TG. Similar ISH staining patterns were seen in both DRG and TG human samples. Arrows identify neurons positive for all three genes of interest. A minimum of 200 cells per ganglion per human sample was counted.

To examine why histamine-induced sensory activation results in less scratching behavior in the cheek compared to the nape of the neck, we integrated ISH with functional ELISA assays to evaluate differential neuropeptide release/distinct peripheral and central neuronal circuit engagement. At the peripheral level, we observed that histamine induced significantly higher expression of early growth response protein 1 (EGR1) with SP (Fig. 8A–C) and also EGR1+/SST+/SP+ (6–7-fold changes) in TG compared to DRG. Functionally, we found that primary cultured TG sensory neurons release significantly more SP than primary cultured DRG sensory neurons following incubation with histamine (*p = 0.0265) (Fig. 8G). At the central level, TNC has a significantly larger population of neurons expressing TacR1 (receptor for SP), Npr1 (receptor for BNP), and the immediate early gene cFos as compared to the spinal dorsal horn (Fig. 8D–F). Interestingly, we found the 50% reduction of Npr1+/cFos+ in the TNC compared to the spinal dorsal horn. These results suggest that the trigeminal system transforms an itch signal into a nociceptive signal through this Npr1/TacR1 convergence at the higher center.

Fig. 8 — A Representative in situ hybridization images of EGR1, SST, and SP in the DRG of histamine (i.d. 100 µg/20 µL). B Representative in situ hybridization images of EGR1, SST, and SP in the TG of histamine (i.d. 100 µg/20 µL). C There is a higher percentage of TG neurons that stained for EGR1/SST/SP as compared to the DRG. D Representative in situ hybridization images of cFos, NPR1, and TacR1 in the spinal dorsal horn. E Representative in situ hybridization images of cFos, NPR1, and TacR1 in the TNC. F There is a higher percentage of Sp5C neurons that stained for cFos/NPR1/TacR1 as compared to the Spinal Dorsal horn. G Primary culture TG neurons release significantly more SP as compared to DRG when exposed to 50 µM Histamine in vitro. H Cartoon schematic of the data presented in this work illustrates that Histamine and Chloroquine activate an overlapping population of SP and SST in the TG to produce a mouse model of pain and itch responses. Data is presented as Mean ± Standard Deviation. Each dot represents one biological replicate. ISH data was seen in at least 2 biological replicates. Scale bar = 50 µm. A minimum of 150 cells was counted per ganglion or spinal region. Created in BioRender; https://BioRender.com/gswovim.

Discussion

Our findings suggest a regional variation in itch response between the cheek and dorsal nape (Figs. 1 and 8G). These differences do not appear to be explained by variations in innervation density (Fig. 2) but may instead relate to distinct populations of sensory neurons expressing specific neuropeptides and receptors (Figs. 3 and 4). The divergence in facial histaminergic and chloroquine-dependent itch processing appears to be associated with the specific proportion of TG neurons co-expressing Substance P (SP) with Sst, MrgprA3, Hrh1, and Hrh3 (Figs. 5–7) and higher expression of Egr1/Sst/Sp as compared to the DRG. Furthermore, our pharmacological data (Fig. 4) point toward HRH3 as a potential transducer of histamine-induced pain in addition to itch. These histological patterns were also observed in human DRG and TG samples (Fig. 7), suggesting translational relevance. At the circuit level, histamine activation of receptors expressed within MrgprA3-positive neurons likely contributes to nocifensive wiping in the cheek, conclusions supported by pharmacological data (Fig. 4) and SP release in the TG compared to DRG (Fig. 8G). Additionally, a higher percentage of co-expression of cFos in Npr1+ and TacR1+ cells in TG compared to DRG provides evidence that intradermal histamine recruits SP-expressing TG sensory neurons, which may trigger central SP release and activate Npr1+/TacR1+ expressing TNC interneurons (Fig. 8D–F). Our study indicates that facial itch processing is molecularly and behaviorally distinct from the body; while body itch (DRG) has been modeled on a relatively segregated population, the TG may utilize a more integrated system where SP-mediated synergy plays a role in the itch-pain switch.

We found a distinct neuropeptide signature in the TG that supports our claim of regional specificity. Our data show that, unlike the DRG, a significantly higher percentage of itch-related neurons expressing HRH1 or MrgprA3 in the TG co-express both SST and Substance P (SP) (Figs. 5 and 6). This finding is crucial because it provides molecular evidence for a more complex peripheral coding system in the TG. While it’s well-established that glutamate is the major neurotransmitter for all primary sensory neurons³⁴, it’s the co-expressed neuropeptides that provide a contextual code, or label, for the central nervous system to interpret the signal as itch or pain^20,21,28. This dual neuropeptide expression in the TG provides a basis for the different behavioral outcomes we observed in the face, which is consistent with the report that low-dose capsaicin provokes scratching, whereas higher doses induce wiping^16,19.

This unique molecular signature in the TG suggests a circuit that can process both itch and pain signals. First, we demonstrate significantly increased expression of SST+/SP+ neurons, which also express EGR1, a marker of DRG and TG neuronal activation (Fig. 8). Further, we demonstrate with ELISA that primary culture TG releases significantly more SP when exposed to histamine (Fig. 8G). We also demonstrate that intradermal injection of histamine triggers induction of cFos in Npr1+ and TacR1+ TNC interneurons, bolstering our hypothesis that our SST+/SP+TG neurons, upon reaching the TNC, engage with central circuits in a way that is distinct from the DRG to dorsal horn. We propose a circuit in which these TG neurons act as the initial signal, differentially engaging downstream interneurons to transmit either an itch or a pain signal. While the Nppb-GRP-GRPR signaling axis is well-characterized in the spinal cord, our data suggests a parallel mechanism within the trigeminal system for cheek-directed itch. The finding that GRPR-expressing neurons, the downstream targets of the NPR1-excitatory interneuron relay, can function as projection neurons^35,36 is particularly relevant to the TNC, suggesting that the H1/H3-mediated synergistic signal originates in the TG and is processed through a TNC gate, where GRPR+ projection neurons may then relay the integrated itch-pain information directly to higher-order centers like the parabrachial nucleus.

An unexpected finding was the presence of a population of NPR1+/TacR1+ interneurons, which exists in the TNC but is not found in the spinal dorsal horn, and which are activated following intradermal histamine administration (Fig. 8D–F). How these interneurons might differentiate between itch versus pain sensations is an area that requires further investigation, especially given that there is evidence of TacR1+ neurons functioning as spinal projection neurons required for transmitting itch sensations from the spinal dorsal horn to the parabrachial nucleus^35,37 and contain another subset that mediate pain, as is seen in the spinal dorsal horn³⁸.

Our findings on the co-expression of Substance P with other itch-related markers warrant a more nuanced discussion of its role. While we initially focused on the association of SP with pain, it is now clear from a substantial body of literature, including the work by Azimi et al., that SP also plays a significant role in itch signaling, particularly through its actions on Mas-related G protein-coupled receptors on peripheral skin mast cells and sensory neurons³⁹. Our finding that a high percentage of HRH1-positive and HRH3-positive neurons co-express SP suggests that SP may not only transmit pain signals but may also contribute to the switch from itch to pain, possibly by activating a separate pain circuit. The finding that the HRH3-specific agonist, immethridine, induced wiping rather than scratching further supports this idea. This histamine-triggered SP release (Fig. 8F) may activate a previously identified monosynaptic facial pain circuit^40,41, accounting for the increase in pain following intradermal histamine injection.

A major limitation in this study is that we used global knockout mice and pharmacological inhibition. Since SP and SST are expressed in other neuronal tissues, removing these genes globally does mean that our results could be due, in part or entirely, to the loss of SP or SST in the spinal cord, spinal trigeminal nucleus, or brain. Similarly, our TacR1 antagonist, L-733,060, has been demonstrated to cross the blood–brain barrier²⁹, meaning that our change in itch behavior could be due to antagonism of TacR1 in the spinal cord, spinal trigeminal nucleus, or brain. JS-11 also has limited ability to cross the blood–brain barrier³¹, meaning that our reductions in itch in JS-11-treated mice could be due to antagonism of NPR1 in the brain, as opposed to antagonism of NPR1 in the spinal cord and trigeminal nucleus.

We demonstrated a correlation between the unique neuropeptide signature and behavioral outcomes. Future work using cell-specific targeting and optogenetic techniques will be essential to establish causality and to fully elucidate the complete nociceptive pathway. This future research should also investigate the specific types of central neurons our identified TG neurons connect with, specifically addressing whether they synapse with labeled line or polymodal projection neurons, a key point highlighted by Moser and Giessler in their work on the nucleus caudalis⁴².

Next, our findings on the distinct expression of facial itch may have significant clinical implications for conditions that disproportionately affect the head and face. The unique neuropeptide signature we identified in the trigeminal ganglia (TG), particularly the co-expression of Substance P (SP) and Somatostatin (SST), suggests that the neural pathways for facial itch (Fig. 8H) are fundamentally different from those in the body and may explain conditions like trigeminal neuralgia, shingles, and facial atopic dermatitis are often resistant to standard treatments and are characterized by a mixture of both itch and pain.

In summary, our study highlights the importance of regional differences in sensory processing by identifying distinct neuron subpopulations in the TG (Fig. 8H). Our findings provide a critical foundation and a clear rationale for future studies that can directly investigate the central circuits and their synaptic plasticity in the TNC, with significant implications for understanding and treating itch disorders of the face.

Materials and methods

Systematic review

To perform our systematic review of itch literature to determine if other researchers had reported differences in the amount of scratching seen in the dorsal nape and cheek, PubMed was searched from 2007 through 2022 with the key words “ITCH” or “PRURITUS” and “NAPE”, “DORSAL NAPE”, or “CHEEK”. Also included were the terms “HISTAMINE”, “CHLOROQUINE”, “SLIGHRL-NH2” or “PAR-2”, “SEROTONIN”, “AYPGKF-NH2” or “PAR-1”, “IL-31” or “INTERLEUKIN 31”. Cathepsin E data is from an article that used another pruritogen as a control. Articles not perform research in mice were excluded from analysis. Articles were only included if they performed intradermal (i.d.) injections. In articles with multiple doses, the highest dose was used. Additionally, in articles using wild-type and knockout mice, data from only wild-type mice were included in our analysis. Dosing was generally consistent across articles: histamine, serotonin, SLIGRL-NH₂, AYPGKF-NH₂, and chloroquine used 90–120 μg per injection. IL-31 used 30 μg for dosing. Since there is no set duration for studying scratching behavior, papers found scored scratching behavior for any duration between 15 min up to 60 min; therefore, the number of scratching bouts was normalized to the “# scratching bouts/min” for comparison. This was achieved by determining the length of the y-axis in pixels and the length in pixels of the mean value of the bar or the distance between the x-axis and the centroid point in line graphs in these manuscripts. The y-axis in all articles found corresponds to the number of scratching bouts counted during those references’ recording period. To calculate the “# scratching bouts/min”, the pixel value height of the bar graph or distance from the x-axis was divided by the total length of the y-axis.

Chemicals

Histamine was ordered from Sigma Aldrich (Catalog # H7125). Wheat Germ Agglutinin conjugated to Alexa Fluor 350 was ordered from Invitrogen (Catalog # W11263). L-733,060 (Catalog # 1145), 2-Pyridylethylamine (Catalog # 2478), and immethridine (Catalog # 2315) were ordered from Tocris. JS-11 (1-cyclohexyl-3-(cyclopropylmethyl)-N-((3-methylisoxazol-5yl)methyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide) was custom synthesized by ChemBridge. The HRH1 antagonist, Diphenydramine (Catalog # 3072), and the HRH3 antagonist JNJ-5207852 (Catalog # 4020) were purchased from Tocris.

Animals

All mice were housed at NC State University, and all experiments were IACUC approved (protocols 22-167-B, 19 -047-B, and 16-038-B). We have complied with all relevant ethical regulations for animal use. Mice were given food and water ad libitum and kept on a 12-h dark cycle (6 am–6 pm EST). All inbred mouse lines were bred in-house from mice obtained from The Jackson Laboratory or the NIH. The following lines were purchased from the Jackson Laboratory: C57Bl6/J (Strain # 000664), B6.Cg-Tac1^tm1Bbm/J (Substance P (SP) KO) (Strain # 004103)²¹, Sst^{tm2.1(cre)Zjh}/J (SST-cre) (Strain # 013044)⁴², B6;129S-Tac1^{tm1.1(cre)Hze}/J (SP-cre) (Strain # 021877)⁴³, and B6.CgGt(ROSA)26Sor^{tm9(CAG0tdTomato)Hze}/J (Ai9) (Strain # 007909)⁴⁴. BNP-KO mice were a generous gift from Dr. Mark Hoon (NIH)²¹. Crl:CD-1(ICR) (CD-1) mice were ordered from Charles River Laboratories (Strain # 022). SST-cre::Ai9 and SP-cre::Ai9 mice were generated by crossing the SST-cre or SP-cre and Ai9 lines. MCPT5-cre mice were a generous gift from Dr. Axel Roers, Technical University, Darmstadt, Germany, and were crossed with B6.129P2-Gt(ROSA)26Sor^tm1(DTA)Lky/J (RSOA^DTA) (Strain# 009669) mice⁴⁵ purchased from the Jackson laboratory to generate MCPT5-cre::DTA mice.

Scratching behavior

Histamine injection: All mice received intradermal injections of 100 µg/20 µL histamine in sterile 1× phosphate-buffered saline (1× PBS). This concentration and volume were used for all intradermal injections. Immethridine and 2-Pyridylethylamine were intradermally injected with the same 100 µg/20 µL concentration. Mice were injected in either Dorsal Nape¹¹ or Cheek¹³. L-733,060 was dissolved in sterile 1× PBS, and 50 µL of a 1 mg/kg solution was administered intravenously 30 min prior to histamine injection^29,30. JS-11 was dissolved in DMSO, and the injection solution consisted of 10% Tween-80, 20% DMSO, and 70% Normal saline. A total of 16.3 µg/50 µL of JS-11 was administered intraperitoneally 30 min prior to histamine injection³¹. The same mice were used for both the Cheek and Nape tests. For drug administration injections, mice were randomly assigned to receive Vehicle, L-733-060, orJS-11. Mice were then randomized to the injection site. After a 10-day washout period, mice were treated with vehicles, then treated with JS-11 or L-733,060, and mice treated with JS-11 or L-733,060 were treated with vehicles. Diphenhydramine (10 mg/kg)⁴⁶ and JNJ-5207852 (10 mg/kg)⁴⁷ were administered intraperitoneally 30 min prior to the injection of histamine into the right cheek. For all experiments, mice were recorded for 30 min post injection. Chloroquine (CQ) was injected at a concentration of 100 µg/20 µL into the dorsal nape or the cheek of C57Bl6 or CD-1 mice and recorded for 30 min.

Immunohistochemistry

For Tuj1 antibody staining, DRG and TG were isolated from SST-cre::Ai9 mice, fixed in 4% PFA for 24 h at 4 °C, cryoprotected using a 30% sucrose solution incubation (~72 h at 4 °C), and then frozen in OTC media. Sections were cut at 14 µm and placed in 4% PFA for 15 min at room temperature. Slides were then blocked with a solution of 5% BSA with 0.1% Triton-X100. Rabbit Anti-mouse Tuj (Abcam, Catalog # ab18207) (1:500 in 5% blocking solution) was applied to sections, which were then incubated overnight at 4 °C. After 16–24 h, the slides were washed in ice-cold 1× PBS three times and then incubated for 45 min at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher, catalog # A-11008). Samples were imaged on a Leica DM500B epifluorescent microscope. For PGP9.5 staining, skin from the cheek and dorsal nape was collected after depilation with Nair. The tissue was then fixed in 4% paraformaldehyde (PFA) for 24 h at 4 °C, followed by cryoprotection in a 30% sucrose solution for approximately 72 h at 4 °C. The samples were then embedded and frozen in OCT (Optimal Cutting Temperature) compound. Immunohistochemistry was performed on the frozen sections. A primary antibody, rabbit antiPGP9.5 (Proteintech, Catalog #14730-1), was diluted to 1:250 in 5% blocking solution and applied to the sections. The sections were then incubated overnight at 4 °C. Following incubation, slides were washed three times with ice-cold 1× PBS. A secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher, Catalog #A-11008), was then applied and incubated for 2 h at room temperature. The samples were imaged using a Leica Stellaris confocal microscope with a white light laser source.

Skin innervation tracing

Mice were briefly anesthetized with isoflurane prior to injection of wheat germ agglutinin (WGA) that was conjugated to Alexa Fluor 350 (Invitrogen, Catalog # W11263) (WGA-350) to determine changes in the population sizes of skin-innervating DRG and TG neurons⁴⁸. WGA-350 was injected intradermally into both cheeks and the dorsal nape. Both TG and C3-T2 DRG were isolated 24 h after injection⁴⁸. TG and DRG were then placed in OCT media and quickly frozen on dry ice and stored at −80 °C for up to 3 months. Cheek injections were bilateral to obtain the greatest potential number of TG sensory neurons that innervate the skin.

Fluorescent in situ hybridization

In situ hybridization was carried out using the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, Catalog # 323100). DRG and TG were isolated from 8-week-old mice 24 h after receiving 50 µL of a 1 mg/mL solution of Alexa Fluor-conjugated Wheat Germ Agglutinin (WGA-350) and frozen in OTC media at −80 °C within 30 min. These sections were cut at a thickness of 10 µm and placed onto Superfrost Gold Plus Slides (Fisher, cat. # 15-188-48). After sectioning, sections were air-dried at −20 °C for 2 h. After this, slides were placed in 4% paraformaldehyde (4% PFA) made with DEPC water (Thermo Fisher, Catalog # 750023) at 4 °C for 15 min. Slides were then washed twice in 1× PBS made with DEPC water. Following this wash step, slides were dehydrated at room temperature in 50% ethanol for 5 min, 70% ethanol for 5 min, and two washes in 100% ethanol for 5 min. The 50% ethanol and 70% ethanol solutions were made with DEPC water. The slides were then kept at −20 °C in 100% ethanol overnight. Ethanol was purchased from Millipore Sigma (Catalog # 459836-2L).

The following day, the slides were removed from 100% ethanol and allowed to air-dry at room temperature for 5 min. A hydrophobic barrier was drawn around the samples using a hydrophobic barrier pen (ACD, Catalog # 310018). The hydrophobic barrier was allowed to dry for 5 min. After drying, roughly 5 drops of RNAscope hydrogen peroxide were added to fill the area within the hydrophobic barrier on the slide and incubated for 10 min at room temperature. The slides were then washed in DEPC-treated water. After this wash, roughly 5 drops of RNAscope Protease III were added to fill the area within the hydrophobic barrier and incubated for 30 min at room temperature. The slides were then washed twice in 1× PBS made with DEPC water. Sections were hybridized on the same day; therefore, sodium citrate was not used.

For hybridization, the following probes were ordered from ACD: Human TAC1 (Substance P) (Catalog# 310711-C3), human SST (Catalog # 310591-C2), human HRH1 (Catalog # 416501), human HRH3 (Catalog # 402191), mouse Tac1 (Catalog # 410351- C2), mouse SST (Catalog # 404631-C3), mouse Hrh1 (Catalog #491141), mouse Hrh3 (Catalog # 428481), mouse Npr1 (Catalog # 484531-C3), mouse TacR1 (Catalog # 428781), mouse EGR1 (Catalog # 423371), and mouse Fos (Catalog # 316921-C2). All DRG and TG slides were stained with the corresponding species-specific probes (human or mouse). All slides were stained with SST and Tac1, and either Hrh1 or Hrh3. To hybridize the probes, the hydrophobic barrier area was filled with ~6 drops of the appropriate probe mixture and then incubated for 2 h at 40 °C in a HybEZ oven. After this incubation, slides were washed in 1× wash buffer twice for 2 min (each wash). For the first two amplification steps, ~6 drops of RNAscope Multiplex FL v2 Amp1 or Amp2 were added to the area within the hydrophobic barrier and incubated for 30 min at 40 °C. For the third hybridization step, RNAscope Multiplex v2 Amp3 was added to the area within the hydrophobic barrier and incubated for 15 min at 40 °C. After each hybridization step, the slides were washed twice in 1× wash buffer for 2 min at room temperature.

To develop the HRP signal, the following steps were performed: roughly 6 drops of RNAscope Multiplex FL v2 HRP-C1, HRP-C2, or HRP-C3 were added to the area within the hydrophobic barrier on the slide and incubated for 15 min at 40 °C. Slides were then washed twice for 2 min in 1× wash buffer at room temperature. To the area in the hydrophobic barrier 200 µL of Opal 520 (channel 1) (Akoya Biosciences, Catalog # FP1487001KT), Opal 570 (channel 2) (Akoya Biosciences, Catalog # FP1488001KT), or Opal 690 (channel 3) (Akoya Biosciences, Catalog # FP1497001KT) was added, and the slides were incubated at 40 °C for 30 min. All dyes were diluted 1:1000. After incubation, slides were washed twice in 1× wash buffer for 2 min at room temperature. Finally, roughly 6 drops of RNAscope Multiplex FL v2 HRP blocker were added to the area within the hydrophobic barrier and incubated at 15 min at 40 °C. After this incubation, the slides were washed twice in 1× wash buffer for 2 min at room temperature.

Cell counting and quantification

Images were collected on a Leica DMB500 epifluorescence microscope. Cells were counted based on appearance since DRG and TG neurons have distinctly round to ovoid shapes with a large, round central gap where the nucleus is located. Skin-innervating neurons were normalized to the total number of WGA-350 neurons. In Figs. 5 and 7, cell populations were normalized to the total number of SST+ cells counted. In Fig. 2, the percentage of SST-expressing neurons was normalized to the total number of TUJ-positive neurons counted. In Fig. 6, cell populations were normalized to the total number of WGA-350 neurons counted. In Fig. 8, cell populations were normalized to the total number of EGR1+ cells or cFos+ cells counted. Cells were defined as >10 puncta surrounding or occluding the DAPI signal. A minimum of 300 neurons was counted per mouse per ganglia or spinal region, and a minimum of 200 neurons was counted per human per ganglia. Intraepidermal nerve fiber images were acquired using a Leica DMB epifluorescent microscope at 40× magnification. For each mouse, 3 sections were analyzed, and 3 fields of view were captured from each section. Intraepidermal nerve fibers were counted manually using ImageJ software. Nerve fiber density was calculated as the number of fibers per millimeter of epidermal length.

Substance P (SP) ELISA

Primary sensory neurons were isolated from the dorsal root ganglia (DRG) and trigeminal ganglia (TG) and placed in ice-cold Neurobasal A media (Thermo Fisher, 10888022) supplemented with PenStrep (Thermo Fisher, 15140122), Glutamax (Thermo Fisher, 35050061), and B27 (Thermo Fisher, 17504044). Respective ganglia were then digested using a mixture of dispase-collagenase (5 mg collagenase (MPBio, 02150704-CF) in 500 µL HBSS (VWR, 02-0121-0500) and 500 µL of Dispase (Stemcell Technologies, 07913)) in the water bath set at 37 °C for 1 h, with mixing every 15–20 min by gently flicking the tube. The ganglia were further dissociated using a glass Pasteur pipette (20–30 times) to obtain a single-cell suspension. The cells were then washed in 10 mL of neurobasal media and centrifuged for 15 min at 1000 rpm. The resulting cell pellet was resuspended in 500 µL of neurobasal media, counted, and plated at a density of 12,000 cells per well of a 24-well plate coated with 15 µg/mL poly-L-ornithine (Sigma, P3655) and 100 µg/ml laminin (Sigma, L2020). Wells with and without cells were stimulated with either 50 μM histamine (Sigma, H7125) or vehicle (DMEM/PBS). Cells were incubated for 15 min at 37 °C with 5% CO₂. After this incubation, the supernatant was collected and stored at –80 °C immediately. The amount of SP released from these cells was measured using the Substance P ELISA kit (Abcam, ab288318).

Statistical analysis

Data was analyzed in GraphPad Prism (version 10.0.0 or higher). Paired 2-tailed t-tests were performed to compare histamine-induced scratching in the cheek versus the dorsal nape. Region-based data was analyzed with a 2-way ANOVA with a post hoc Holm – Šídák test for multiple comparisons. Other data was compared with a 1-way ANOVA with a post hoc Holm – Šídák test for multiple comparisons. Literature data was compared using an unpaired 2-tailed Mann–Whitney U-test. For all tests, the initial significance value was set to α = 0.05.

Supplementary information

42003_2026_10128_MOESM1_ESM.pdf^{(27.5KB, pdf)}

Description of Additional Supplementary Files

Supplementary Data^{(70.3KB, xlsx)}

Acknowledgements

The authors would like to thank Dr. Mark Hoon for critically reviewing the manuscript and providing excellent feedback. We thank Dr. Axel Roers for providing this Mcpt5-cre line that we have been breeding in-house for a neuro-immune itch project. The authors would like to thank Megan Cherry and Morgan Devers for their help in quantifying the total scratching time in mice following chloroquine injection. This work was supported by NIH grant R01AR077692, intramural NCSU funding, and a gift provided by Dr. John M. Davis.

Author contributions

Ideation – S.K.M.; Conceptualization –S.K.M., J.J.W.; Methodology – J.J.W., T.O.; Software – Not Applicable; Validation – J.J.W., N.W.; Formal Analysis – J.J.W.; Investigation – J.J.W., N.W., A.V.; Data Curation – J.J.W.; Writing- Original Draft – J.J.W.; Visualization – J.J.W., S.K.M.; Writing – Review & Editing – J.J.W., S.K.M., T.O., B.D.X.L.; Project Administration, Resources & Funding Acquisition – S.K.M.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Benjamin Bessieres.

Data availability

Data used to generate graphs can be found in the supplementary file associated with this document. Other data will be made available by the corresponding author upon request.

Code availability

GraphPad Prism (version 10.0.0 or higher) was used for data analysis. No custom code was generated for the analysis of this data.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-10128-9.

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47.Rossbach, K. et al. Histamine H1, H3 and H4 receptors are involved in pruritus. Neuroscience190, 89–102 (2011). [DOI] [PubMed] [Google Scholar]
48.Buchholtz, L. J. von, Lam, R. M., Emrick, J. J., Chesler, A. T. & Ryba, N. J. P. Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning. Pain. 161, 2212 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

42003_2026_10128_MOESM1_ESM.pdf^{(27.5KB, pdf)}

Description of Additional Supplementary Files

Supplementary Data^{(70.3KB, xlsx)}

Data Availability Statement

Data used to generate graphs can be found in the supplementary file associated with this document. Other data will be made available by the corresponding author upon request.

GraphPad Prism (version 10.0.0 or higher) was used for data analysis. No custom code was generated for the analysis of this data.

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[CR22] 22.Sun, Y.-G. & Chen, Z.-F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature448, 700–703 (2007). [DOI] [PubMed] [Google Scholar]

[CR23] 23.Wheeler, J. J., Allen-Moyer, K. N. & Mishra, S. K. A systematic review of animal models and sex as a variable in itch research. Itch. 5, e40 (2020). [Google Scholar]

[CR24] 24.Laborc, K. F. et al. Trigeminal activation patterns evoked by chemical stimulation of the dura mater in rats. J. Headache Pain.21, 101 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Bogusch, G. Innervation of the dermatomes in the neck of the mouse. Acta Anat.129, 275–278 (1987). [DOI] [PubMed] [Google Scholar]

[CR26] 26.Solinski, H. J. et al. Nppb neurons are sensors of mast cell-induced itch. Cell Rep.26, 3561–3573.e4 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Day, I. N. Molecular analysis of two general markers of neurons and the diffuse neuroendocrine system: neuron-specific enolase and PGP9.5. Biochem. Soc. Trans.16, 454–457 (1988). [DOI] [PubMed] [Google Scholar]

[CR28] 28.Cao, Y. Q. et al. Primary afferent tachykinins are required to experience moderate to intense pain. Nature392, 390–394 (1998). [DOI] [PubMed] [Google Scholar]

[CR29] 29.Varty, G. B. et al. The gerbil elevated plus-maze II: anxiolytic-like effects of selective neurokinin NK1 receptor antagonists. Neuropsychopharmacology27, 371–379 (2002). [DOI] [PubMed] [Google Scholar]

[CR30] 30.Rupniak, N. M. J., Carlson, E., Boyce, S., Webb, J. K. & Hill, R. G. Enantioselective inhibition of the formalin paw late phase by the NK1 receptor antagonist L-733,060 in gerbils. Pain. 67, 189–195 (1996). [DOI] [PubMed] [Google Scholar]

[CR31] 31.Solinski, H. J. et al. Inhibition of natriuretic peptide receptor 1 reduces itch in mice. Sci. Transl. Med.11, eaav5464 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Durant, G. J., Ganellin, C. R. & Parsons, M. E. Chemical differentiation of histamine H1- and H2-receptor agonists. J. Med. Chem.18, 905–909 (1975). [DOI] [PubMed] [Google Scholar]

[CR33] 33.Kitbunnadaj, R. et al. Identification of 4-(1H-imidazol-4(5)-ylmethyl)pyridine (immethridine) as a novel, potent, and highly selective histamine H(3) receptor agonist. J. Med. Chem.47, 2414–2417 (2004). [DOI] [PubMed] [Google Scholar]

[CR34] 34.Wanaka, A. et al. Glutamate-like immunoreactive structures in primary sensory neurons in the rat detected by a specific antiserum against glutamate. Exp. Brain Res.65, 691–694 (1987). [DOI] [PubMed] [Google Scholar]

[CR35] 35.Sheahan, T. D., Warwick, C. A., Fanien, L. G. & Ross, S. E. The neurokinin-1 receptor is expressed with gastrin-releasing peptide receptor in spinal interneurons and modulates itch. J. Neurosci.40, 8816–8830 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Polgár, E. et al. Grpr expression defines a population of superficial dorsal horn vertical cells that have a role in both itch and pain. Pain. 164, 149–170 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Akiyama, T. et al. A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch. Pain. 156, 1240–1246 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Barik, A. et al. A spinoparabrachial circuit defined by Tacr1 expression drives pain. eLife10, e61135 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Azimi, E. et al. Substance P activates Mas-related G protein-coupled receptors to induce itch. J. Allergy Clin. Immunol.140, 447–453.e3 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Ding, Y. Q., Takada, M., Shigemoto, R. & Mizuno, N. Trigeminoparabrachial projection neurons showing substance P receptor-like immunoreactivity in the rat. Neurosci. Res.23, 415–418 (1995). [DOI] [PubMed] [Google Scholar]

[CR41] 41.Rodriguez, E. et al. A craniofacial-specific monosynaptic circuit enables heightened affective pain. Nat. Neurosci.20, 1734 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Taniguchi, H. et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron71, 995–1013 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Harris, J. A. et al. Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation. Front. Neural Circuits8, 76 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci.13, 133–140 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Voehringer, D., Liang, H.-E. & Locksley, R. M. Homeostasis and effector function of lymphopenia-induced ‘memory-like’ T cells in constitutively T cell-depleted mice. J. Immunol.180, 4742–4753 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Sugimoto, Y., Umakoshi, K., Nojiri, N. & Kamei, C. Effects of histamine H1 receptor antagonists on compound 48/80-induced scratching behavior in mice. Eur. J. Pharmacol.351, 1–5 (1998). [DOI] [PubMed] [Google Scholar]

[CR47] 47.Rossbach, K. et al. Histamine H1, H3 and H4 receptors are involved in pruritus. Neuroscience190, 89–102 (2011). [DOI] [PubMed] [Google Scholar]

[CR48] 48.Buchholtz, L. J. von, Lam, R. M., Emrick, J. J., Chesler, A. T. & Ryba, N. J. P. Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning. Pain. 161, 2212 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Substance P and somatostatin neurons limit facial itch by recruiting distinct nociceptive circuits in the brainstem

Joshua J Wheeler

Nidha Williams

Aditi Vigneshwar

B Duncan X Lascelles

Thierry Olivry

Santosh K Mishra

Abstract

Introduction

Results

Regional scratching responses differ between the face and neck

Fig. 1. Scratching behavior differs significantly between the dorsal nape and cheek of inbred and outbred mice.

Regional itch variations are not explained by peripheral innervation densities

Fig. 2. The cheek is more densely innervated by SST-positive neurons than the dorsal nape.

Distinct role of SP in modulating itch response in the cheek and dorsal nape

Fig. 3. Loss of Substance P significantly increases the total number of ipsilateral cheek-directed scratching bouts.

Histamine receptor-specific itch induction

Fig. 4. Specific activation of HRH3 with immethridine induced significantly fewer scratching bouts and significantly higher wiping bouts.

Regional variations in sensory neuron populations underlying itch and pain

Fig. 5. There is a significant increase in neurons that express both SST and SP in the TG of male mice.

Fig. 6. Skin-innervating TG sensory neurons have larger populations of neurons positive for HRH1, SST, and SP; HRH3, SST, and SP; or MrgprA3, SST, and SP as compared to skin-innervating DRG sensory neurons.

Fig. 7. Human DRG and TG recapitulate the changes in DRG and TG sensory neuron populations of HRH1 or HRH3, SST, and SP.

Fig. 8. Trigeminal (TG) pathways show peripheral activation and neuropeptide involvement in response to Histamine than Dorsal Root Ganglion (DRG) pathways and central engagement.

Discussion

Materials and methods

Systematic review

Chemicals

Animals

Scratching behavior

Immunohistochemistry

Skin innervation tracing

Fluorescent in situ hybridization

Cell counting and quantification

Substance P (SP) ELISA

Statistical analysis

Supplementary information

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Code availability

Competing interests

Footnotes

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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