The Molecular and Cellular Basis of Regional Itch Differences

The Molecular and Cellular Basis of Regional Itch Differences | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Molecular and Cellular Basis of Regional Itch Differences Santosh Mishra, Joshua Wheeler, Nidha Williams, Thierry Olivry This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6466187/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version 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. Here, we investigate the neural basis of regional itch differences, focusing on the role of neuropeptides and histamine receptors expression. Using a combination of behavioral, pharmacological, genetic, histological, and molecular approaches, we identified distinct regional differences in itch response between the cheek and dorsal nape (neck). Our findings reveal that Substance P modulates itch in the cheek, while histamine receptor 1 (HRH1) is the primary receptor mediating itch but not pain in the cheek. We also discovered regional differences in sensory neuron subpopulations, with increased co-expression of HRH1 and neuropeptides in trigeminal ganglion neurons. In brief, we provide new insights into the molecular and cellular mechanisms underlying regional itch differences, highlighting the importance of considering regional variations in itch response to develop effective treatments. Biological sciences/Neuroscience/Sensory processing Biological sciences/Neuroscience/Somatosensory system/Pain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 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 induces a differential behavioral response or differences in itch circuitry in mice is unknown. In humans, the severity of itch is perceived differentially 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 an overlapping population of second-order neurons found in the spinal trigeminal nucleus 15 . In contrast, itch and pain are processed in the DRG sensory neurons and activate separate populations of second-order neurons 19 , 20 . The distinct neural processing of itch in the TG versus the DRG suggests the involvement of specific neuropeptides and signaling pathway involved in transmitting itch signals from the face versus the body. Itch and pain sensations are mediated through specific neuropeptides. Substance P (SP) mediates pain sensations 21 . Meanwhile, itch sensations are mediated by somatostatin (SST)-expressing neurons, which are > 99% co-localized with brain natriuretic peptide (BNP)-expressing neurons 20 , 22 . Activation of these first-order neurons triggers the release of these neuropeptides, which then activate second-order neurons in the dorsal horn of the spinal cord to further process these pain and itch sensations 19 , 20 , 22 , 23 . The significant difference in itch processing between the face (cheek) and torso (neck) reveals a substantial knowledge gap. To address this, we employed a multifaceted approach combining mouse genetics and behavioral, pharmacological, cellular, and molecular techniques. Following intradermal histamine injections into the dorsal nape or cheek, we investigated itch behavior in C57Bl6/J (inbred) and CD-1 (outbred) mice. Additionally, we examined histamine-induced itch behavior in mice lacking BNP or SP as well as in mice treated with antagonists for their respective receptors (NPR1 24 and TACR1). We also assessed nape and cheek skin innervation density by genetically marking somatostatin-expressing neurons with tdTomato. To identify differences in neuronal populations, we used tracing techniques followed by immunohistochemistry and in-situ hybridization to analyze histamine receptors H1 and H3, BNP/SST, and SP in human and mouse DRG and TG. In summary, these experiments aim to elucidate the mechanisms underlying the differences in itch processing between the face and body. RESULTS Regional scratching response between the face and neck We initially performed a literature review of papers published between January 2009 and June 2023 (Fig. 1 B,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, PAR-2 specific agonists (*p = 0.0006) (Fig. 1 B). This data was further confirmed when we combined all scratching bouts/minute values for all pruritogens (*p = 0.0073) (Fig. 1 C). 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. 1 D) 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. 1 E), and no significant differences were seen in the average bout duration (*p = 0.2540) (Fig. 1 F). The time course of histamine-induced scratching bouts differed significantly between the dorsal nape and cheek (Fig. 1 G). Ipsilateral cheek-directed scratching bouts peaked within the first 5 minutes (*p < 0.0001) (Fig. 1 G) and the number of dorsal nape-directed scratching bouts peaked at 15 minutes (*p = 0.0001) (Fig. 1 G). 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. 1 H-K) 26 . Our 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. 1 H &I). Unlike with C57BL/6J mice, bout duration significantly differed between regions in the CD-1 mice (*p = 0.0380) (Fig. 1 J). The time course of histamine-induced scratching bouts differed between regions, with cheek-directed scratching peaking within 5 minutes, and significant differences were observed at 15 (*p = 0.0002), 20 (*p = 0.0477) and 30 minutes (*p = 0.0009) (Fig. 1 K). Overall, our data suggests that regional differences in itch are conserved across outbred and inbred mouse lines, with the cheek being less responsive to histamine-induced itch compared to the dorsal nape. Regional itch differences are not explained by 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 TG 27 and the dorsal nape dermatomes innervated by the DRG 28 . 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 responsible for mediating itch signals induced by histamine 20 , 29 . 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. 2 C - E). 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. 2 F-H). This increase is likely to be responsible for increased SST-positive afferent density between the dorsal nape and the cheek. These findings also establish that the higher innervation density of itch-sensing neurons in the cheek does not correlate with the reduced scratching response observed in this region. 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) 21 , might modulate ipsilateral cheek-directed scratching behavior. Based on previous observations where we found the dorsal nape, all SST-expressing DRG sensory neurons are positive for BNP 20 . Further, these BNP-expressing DRG sensory neurons are not positive for Substance P (SP) 22 , a canonical pain-associated neuropeptide. 21 While there is literature about the relative co-expression of these neuropeptides in DRG sensory neurons, there is no information about their co-expression in TG sensory neurons. 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. 3 A), with no differences in dorsal nape-directed scratching bouts (*p = 0.2390) (Fig. 3 B). We used BNP-KO mice as a control, as these mice should lack an itch 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. 3 E & F). To confirm that our behavior results in SP-KO and BNP-KO mice, we pharmacologically inhibited the SP receptor (TacR1) with L-733,060 31,32 . We found that these mice exhibited significantly higher amounts of ipsilateral cheek-directed scratching bouts than vehicle-treated mice (*p = 0.0198) (Fig. 3 C). 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. 3 D). Mice treated with the NPR1 antagonist JS-11 33 experienced significantly fewer scratching bouts in both the ipsilateral cheek (*p = 0.0338) and the dorsal nape (*p = 0.0396) (Fig. 3 G & 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. 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, while HRH3 is expressed in SP-positive DRG sensory neurons involved in pain 2 . To determine which histamine receptor(s) are responsible for transducing histamine-induced itch, we injected 2-pyridylethylamine (HRH1-specific agonist) 34 and immethridine (HRH3-specific agonist) 35 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. 4 B & C). We found that 2-Pyr induced significantly more cheek wipes as compared to histamine in addition to the cheek directed scratching (*p = 0.0230) (Fig. 4 D). Immethridine also induced more ipsilateral cheek wipes (*p = 0.136) (Fig. 4 D), 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). Our results suggest that HRH1 is the primary histamine receptor mediating itch in dorsal nape-innervating DRG sensory neurons, while HRH3 may play a role in pain-behavior. 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-, 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 behavior 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 , 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. 5 A – D). We further validated this in human using in situ hybridization (ISH; Fig. 5 E-H). Using wheat germ agglutinin (WGA-350) as a neuronal tracer, used here to identify skin-innervating sensory neurons, we also 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. 6 ). Next, we investigated the expression of HRH1 and HRH3 in skin-innervating sensory neurons in mice (Fig. 7 ). Our results showed an increase in the percentage of TG sensory neurons expressing HRH1, SST, and SP, as well as HRH3, SST, and SP, compared to the DRG. We further corroborated these findings in human TG versus DRG regional-specific differences in human sensory neurons (Fig. 8 ). DISCUSSION Our study reveals significant regional differences in itch response between the cheek and dorsal nape (Fig. 1 ). These differences are not attributed to variations in innervation density (Fig. 2 ), but to distinct populations of sensory neurons expressing specific neuropeptides and receptors (Fig. 3 – 4 ). Here, we demonstrate that the difference in facial histaminergic itch processing is due to differences in the proportion of TG neurons that co-express Substance P with SST, HRH1, and HRH3 (Figs. 5 , 6 & 7 )). Behaviorally, this results in the likely activation of HRH1, inducing pain responses in the cheek. Further, we demonstrate that HRH3 might transduce histamine activation into pain (Fig. 4 ). Our histological results were also recapitulated in human DRG and TG samples (Figs. 5 & 8 ), extending their translational relevance. Finally, our results indicate that the TG processes facial itches fundamentally differently by a mechanism similar to what is seen in the gate control model of itch and pain processing (Fig. 9 ). Here, we confirmed that the number of histamine-induced ipsilateral cheek-directed scratching bouts is significantly reduced as compared to the number of histamine-induced dorsal nape scratching bouts by systematically comparing the scratching bouts by using the C57Bl/6J mice and the same amount of histamine in the same injection volume for both the cheek and nape injections. These results confirm that the reduction in the total number of ipsilateral cheek-directed scratching bouts is due to a difference in the neurophysiology of the cheek skin as opposed to differences in injection volumes or dose (at least for histamine). Interestingly, outbred CD-1 mice, which are thought to be a better model for human research 36 , had significantly fewer ipsilateral cheek-directed scratching bouts, corroborating the results from the inbred line. Furthermore, CD-1 mice have significantly shorter scratching bout durations (Fig. 1 I), implying that they also perceive histaminergic itch in the cheek as less intense than histaminergic itch in the dorsal nape. Validation of regional differences in histamine-induced itch between inbred and outbred lines suggests that the study findings are not limited to a specific mouse strain and likely translatable to humans. One of the limitations of our study is that we specifically investigated the role of histamine in regional itch differences, despite literature suggesting that other itch mediators, such as chloroquine, serotonin, SLIGRL-NH2, IL31, and proteases, may also exhibit similar regional differences (Fig. 1 ). There are mostly three reasons for this focus: Firstly, histamine is a well-known endogenous mediator for itch. Further, its involvement in itch processing is a helpful model for understanding the neural mechanisms underlying regional differences itch sensitivity. Secondly, in sensory neurons, histamine's-induced itch primarily mediated by the histamine receptor 1 (HRH1). This receptor specificity allows for targeted investigation of histamine's role in regional itch differences, without the confounding effects of other itch mediators. Lastly, histamine allows for a more in-depth investigation of the neural mechanisms underlying regional itch differences, which can serve as a foundation for future studies on other itch mediators, and the clinical relevance of histamine in itch disorders makes it a prime target for therapeutic intervention at sensory neurons carrying message to the CNS. Histamine-induced behavior hints at TG-specific processing of itch behavior Reports have indicated differences between the DRG and TG at the transcriptome level 3 , 4 , 6 . Yet, the overall assumption has been that itch and pain processing in the TG follows the same pathways as itch processing in the DRG. Electrophysiology work performed by Akiyama et al., 15 further hinted at a difference in pain and itch signaling when they reported that pruritogens and algogens, itch inducers and pain inducers, respectively, results in postsynaptic activation of an overlapping population of neurons in the spinal trigeminal (TG) nucleus. There are currently two main theories about how the nervous system processes itch and pain. The first is a modified label-line theory, which posits that itch and pain are processed by two parallel pathways by the peripheral nervous system and the spinal cord dependent on the input simulus 37 , 38 . Overall, the label line theory is correct for the DRG; SST-positive/BNP-positive neurons primarily process itch, and pain is mediated mainly by SP-expressing DRG neurons 21 , 22 . Further supporting this theory is the finding that activation of the MrgprA3-expressing DRG neurons by capsaicin in TRPV1-KO::MrgprA3 ROSA − TRPV1 mice, wherein TRPV1 is only in MrgprA3-expressing neurons , results in itch – not pain 18 . The second theory of itch and pain processing is the Gate Control theory, which states that separate pain and itch-responding DRG neurons activate the same set of secondary neurons located in the dorsal horn of the spinal cord. This theory was founded by discovery of dynorphin-expressing spinal interneurons activated following TRPV1 or TRPM8 neurons activation 19 . Activation of these dynorphin-expressing interneurons triggers the release of dynorphin, which then suppresses activation of gastrin-related peptide receptor (GRPR)-expressing spinal interneurons, thus preventing the transmission of itch signals 19 . Current research has determined that these GRPR-expressing spinal interneurons are where itch signals converge 19 , 20 , 22 , 23 , 39 , 40 before these interneurons transmit itch signals to the parabrachial nucleus 41 , 42 . This theory is further supported by the finding that SST-dependent activation of SSTR2-subpopulation inhibits pain responses 20 . Both of these theories on itch processing have been assumed to be the de facto pathways by which the TG processes itch behavior despite the lack of evidence, either for or against, the presence of these pathways in the TG nucleus. Our results suggest that the TG itself uses gate control to process facial itch signals despite the unclear presence of these pathways in the spinal trigeminal nucleus 43 . Our mouse data demonstrate that TG have a significantly increased percentage of SST-expressing neurons expressing Substance P compared to the DRG (Fig. 5 ). HRH1, which transduces histamine receptor activation into itch, has significant changes in downstream neuropeptide populations (Figs. 7 & 8 ). In the DRG, HRH1 is predominantly co-expressed with SST, with few neurons being positive for HRH1 and Substance P; however, in the TG, this proportion of HRH1-positive and Substance P-positive neurons roughly triples (Fig. 7 ). This change in HRH1 and Substance P expression also results in a change in behavioral outcomes (Fig. 4 ). HRH3 underwent similar changes in the proportion of neurons positive for Substance P in the TG compared to the DRG (Figs. 7 & 8 ). Interestingly, immethridine, an HRH3-specific agonist 35 , did not induce itch but significantly more ipsilateral cheek-directed wipes than histamine, indicating that histaminergic itch is both receptor-dependent and neuropeptide-dependent. These findings provide a basis for examining how the gate control theory of pain and itch processing may be relevant in this context. The gate control theory of pain and itch processing has evidence that there might be two subtypes: the leaky gate subtype suggesting itch and weak pain signals activate the same pathway 44 , and an intensity coding subtype suggests itch signal transmission is dependent on a frequency or burst of stimulation 45 . While we could not test this hypothesis here, but our results, and those reported in Akiyama et al 15 , alluding to an intensity coding in the TG. Another avenue to explore would be to determine if the histamine-triggered SP-release can activate a previously identified monosynaptic facial pain circuit 46 as these neurons express the receptor for SP 47 . Activation of this pathway by histamine would also likely account for the decrease in itch behavior and increase in pain following intradermal injection of histamine into the cheek. In summary, we identified distinct sensory neuron subpopulations, highlighting the importance of considering regional differences in itch response when designing treatment strategies. Overall, our study has significant implications for the understanding and treatment of itch disorders by developing therapies targeting HRH1 and HRH3 receptors that may provide new treatment options for itch disorders. METHODS 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-Pyrdylethylamine (Catalog # 2478), and immethridine (Catalog # 2315) were ordered from Tocris. JS-11 (1-cyclohexyl-3-(cyclopropylmethyl)-N-((3-methylisoxazol-5-yl)methyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide) was custom synthesized by ChemBridge. 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). Mice were given food and water ad libitum and kept on a 12-hour light dark cycle (6am -6pm 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) 49 , and B6.Cg- Gt(ROSA)26Sor tm9(CAG0tdTomato)Hze /J (Ai9) (Strain # 007909) 50 . BNP KO mice were a generous gift from Dr. Mark Hoon (NIH) 22 . Crl:CD1(ICR) (CD-1) mice were ordered from Charles River laboratories (Strain # 022). SST-cre::Ai9 mice were generated by crossing the SST-cre and Ai9 lines. Itch Behavior Histamine injection: All mice received intradermal injections of 100 µg/20 µL histamine in sterile 1X phosphate buffered saline (1X 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 Nape 11 or Cheek 12 . L-733,060 was dissolved in sterile 1X PBS and 50 µL of a 1mg/kg solution was administered intravenously 30 minutes prior to histamine injection 31 , 32 . 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 minutes prior to histamine injection 33 . The same mice were used for both the Cheek and Nape tests. For drug administration injections, mice were randomly assigned to receive Vehicle, L733-060, orJS-11. Mice were then randomized to the injection site. After a 10-day washout period, mice were treated with vehicle were then treated with JS-11 or L-733,060 and mice treated with JS-11 or L-733,060 were treated with vehicle. Immunohistochemistry For Tuj antibody staining, DRG and TG were isolated from SST-cre::Ai9 mice, fixed in 4% PFA for 24 hours at 4°C, cryoprotected using a 30% sucrose solution incubation (~ 72 hrs at 4°C), and then frozen in OTC media. Sections were cut at 14 µm and placed in 4% PFA for 15 minutes at room temperature. Slides were then blocked with 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 slides, which were then incubated overnight at 4°C. After 16–24 hrs, the slides were washed in ice cold 1X PBS trice and then incubated for 45 minutes at room temperature with Alexa Fluor 488 conjugated goat anti-rabbit IgG (Thermofisher, catalog # A-11008). Samples were images on a Leica DM500B epifluorescent microscope. 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 neurons 52 . WGA-350 was injected intradermally into both cheeks and the dorsal nape. Both TG and C3-T2 DRG were isolated 24 hours after injection 52 . TG and DRG were then placed in OCT media and quickly frozen on dry ice and were 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 hours after receiving 50 µL of a 1mg/mL solution of Alexa Flour-conjugated Wheat Germ Agglutinin (WGA-350) and frozen in OTC media at -80°C within 30 minutes. These sections were cut at 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 hours. After this, slides were placed in 4% paraformaldehyde (4% PFA) made with DEPC water (Thermofisher, Catalog # 750023) at 4°C for 15 minutes. Slides were then washed twice in 1X PBS made with DEPC water. Following this wash step, slides were dehydrated at room temperature in 50% ethanol for 5 minutes, 70% ethanol for 5 minutes, and two washes in 100% ethanol for 5 minutes. 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 minutes. 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 minutes. After drying, roughly 5 drops of RNAscope hydrogen peroxide was added to fill the area within the hydrophobic barrier on the slide and incubated for 10 minutes at room temperature. The slides were then washed in DEPC treated water. After this wash, roughly 5 drops of RNAscope Protease III was added to fill the area within the hydrophobic barrier and incubated for 30 minutes at room temperature. The slides were then washed twice in 1X PBS made with DEPC water. Sections were hybridized and on the same day; therefore, sodium citrate was not used. For hybridization, probes 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), and mouse Hrh3 (Catalog # 428481). 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 hours at 40°C in a HybEZ oven. After this incubation, slides were washed in 1X wash buffer twice for two minutes (each wash). For the first two amplification steps, ~ 6 drops of RNAscope Multiplex FL v2 Amp1 or Amp2 was added to the area within the hydrophobic barrier and incubated for 30 minutes 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 minutes at 40°C. After each hybridization step slides were washed twice in 1X was buffer for 2 minutes 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 minutes at 40°C. Slides were then washed twice for 2 minutes in 1X 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 minutes. All dyes were diluted 1:1000. After incubation, slides were washed twice in 1X wash buffer for 2 minutes at room temperature. Finally, roughly 6 drops of RNAscope Multiplex FL v2 HRP blocker was added to the area within the hydrophobic barrier and incubated at 15 minutes at 40°C. After this incubation, the slides were washed twice in 1X wash buffer for 2 minutes 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 , 8 , and 9 cell populations were normalized to the total number of cells counted. In Fig. 2 , the percentage of SST-expressing neurons was normalized to the total number of TUJ positive neurons counted. Statistical Analysis: Data was analyzed in Graphpad Prism. Paired 2-tailed t-tests were performed to compare histamine-induced scratching in the cheek versus 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. Declarations ACKNOWLEDGEMENTS The authors would like to thank Dr. Mark Hoon for critically reviewing the manuscript and providing excellent feedback. This work was supported by NIH grant R01AR077692, intramural NCSU fundings and gift provided by Dr. John M. Davis. CONFLICTS OF INTEREST The authors declare no conflicts of interest. References Goswami SC et al (2014) Molecular signatures of mouse TRPV1-lineage neurons revealed by RNA-Seq transcriptome analysis. 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Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning. Pain 161:2212 Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6466187","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":448504999,"identity":"0f6fc22e-1011-4759-87d0-a8a506b2b13e","order_by":0,"name":"Santosh Mishra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACHgaGA4wNDAz8UD6ITaQWyQZStICVGRwgVot8z+nEgz933JM3vt38+DMPg43shgMEtBic7d1wmPdMseG2O8fMpHkY0owJa+Hn3XCYsS2BcduNHDZmHobDiQS1yPfzbjj4sy3BfvOMHGagw/4T1sIAdNgB3raExA0SOQxAhx0grMXgzFmgX9oSkmfcSDOTnGOQbDyToMN6cjd/BDrMtn9G8uMPbyrsZPsIOgzNUtKUj4JRMApGwSjAAQAgS0gIAZ2cIgAAAABJRU5ErkJggg==","orcid":"","institution":"North Carolina State University","correspondingAuthor":true,"prefix":"","firstName":"Santosh","middleName":"","lastName":"Mishra","suffix":""},{"id":448505000,"identity":"cb576608-d364-4b2c-b69a-f9d0092b69e0","order_by":1,"name":"Joshua Wheeler","email":"","orcid":"https://orcid.org/0000-0002-1879-1627","institution":"North Carolina State University","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Wheeler","suffix":""},{"id":448505001,"identity":"44433d4d-26d1-48de-9612-3ceba3c541f1","order_by":2,"name":"Nidha Williams","email":"","orcid":"","institution":"North Carolina State University","correspondingAuthor":false,"prefix":"","firstName":"Nidha","middleName":"","lastName":"Williams","suffix":""},{"id":448505002,"identity":"e1a72674-ddcc-4243-bee4-4f6de1ca1c71","order_by":3,"name":"Thierry Olivry","email":"","orcid":"https://orcid.org/0000-0003-1399-0034","institution":"Department of Clinical Sciences, College of Veterinary Medicine, NC State University","correspondingAuthor":false,"prefix":"","firstName":"Thierry","middleName":"","lastName":"Olivry","suffix":""}],"badges":[],"createdAt":"2025-04-16 19:55:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6466187/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6466187/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83445291,"identity":"5246aaf3-1efe-4ccf-9846-f50fde7a21b4","added_by":"auto","created_at":"2025-05-26 10:57:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":132704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScratching behavior differs significantly between the dorsal nape and cheek of inbred and outbred mice. A) \u003c/strong\u003eCartoon highlighting the dorsal nape and cheek injection sites.\u003cstrong\u003e B)\u003c/strong\u003e Results of a literature search for common pruritogens comparing the normalized itch behavior (scratching bouts/minute) of the dorsal nape and cheek reported in the literature.\u003cstrong\u003e C) \u003c/strong\u003eCompiled data from Panel A demonstrating that, regardless of pruritogen, the normalized amount of itch behavior reported is significantly reduced in the cheek as compared to the dorsal nape.\u003cstrong\u003e D) \u003c/strong\u003eIntradermal (i.d.) injection of histamine (100 µg/ 20 µL) induces significantly more scratching bouts in the dorsal nape (innervated by the DRG) than the cheek of C57Bl6/J mice.\u003cstrong\u003e E)\u003c/strong\u003eThere are no significant differences in the total amount of time spent scratching the dorsal nape or cheek following i.d. histamine (100 µg/ 20 µL) in C57Bl6/J mice. \u003cstrong\u003eF) \u003c/strong\u003eThere are no significant differences in scratching bout duration between the dorsal nape and cheek following i.d. histamine (100 µg/ 20 µL) in C57Bl6/J mice.\u003cstrong\u003e G) \u003c/strong\u003eSignificantly more cheek-directed scratching bouts occur within the first 5 minutes following histamine (100 µg/ 20 µL) injection (i.d.), while significantly more dorsal nape-directed scratching bouts occur ~15 minutes following histamine (100 µg/ 20 µL) injection (i.d.).\u003cstrong\u003e H) \u003c/strong\u003eIntradermal (i.d.) injection of histamine (100 µg/ 20 µL) induces significantly more scratching bouts in the dorsal nape than the cheek of CD-1 mice. \u003cstrong\u003eI) \u003c/strong\u003eThere is a significant differences in the total amount of time spent scratching the dorsal nape or cheek following i.d. histamine (100 µg/ 20 µL) in CD-1 mice. \u003cstrong\u003eJ) \u003c/strong\u003eThere are no significant differences in scratching bout duration between the dorsal nape and cheek following i.d. histamine (100 µg/ 20 µL) in CD-1 mice.\u003cstrong\u003e K) \u003c/strong\u003eSignificantly more dorsal nape-directed scratching bouts occur 15 to 25 minutes following histamine (100 µg/ 20 µL) injection (i.d.) in CD-1 mice. Data is presented as Mean ± Standard Deviation. Each dot represents one biological replicate. Significance in Panels A \u0026amp; B were determined using a 2-tailed Mann-Whitney U-test. Significance for Panels C, D, E, G, H, \u0026amp; I were determined using a 2-tailed Student’s t-test. Significance in Panels F \u0026amp; J were determined using a 2-way ANOVA. ** p \u0026lt; 0.01, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/712cbb348be9263fabfba5b7.jpg"},{"id":83445532,"identity":"d206c4d7-ebdb-4965-a01d-a206c381d233","added_by":"auto","created_at":"2025-05-26 11:05:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cheek is more densely innervated by SST-positive neurons than the dorsal nape. A) \u003c/strong\u003eCartoon highlighting the dorsal nape and cheek locations.\u003cstrong\u003e B)\u003c/strong\u003e Cartoon highlighting the location of the TG and DRG.\u003cstrong\u003e C)\u003c/strong\u003eRepresentative confocal images of skin isolated from the dorsal nape of naïve SST-cre::Ai9 mice.\u003cstrong\u003e D)\u003c/strong\u003e Representative confocal images of skin isolated from the cheek of naïve SST-cre::Ai9 mice.\u003cstrong\u003e E) \u003c/strong\u003eQuantification of the innervation density of the dorsal nape and cheek of SST-cre::Ai9 mice. Data is presented as Mean ± Standard Deviation. F) Mouse schematic labeling the locations of the dorsal nape, cheek, DRG, and TG. G) Representative image of SST expression in the TG. H) Representative image of SST expression in the DRG. G) Quantification of SST-positive neurons in the DRG and TG, normalized to Tuj staining. Each dot represents one biological replicate. *p \u0026lt; 0.05 as determined by a paired 2-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/b22c5c9e20d6135b03100ac5.jpg"},{"id":83445292,"identity":"278e6528-cd0e-4128-8e1b-8c4f947982c4","added_by":"auto","created_at":"2025-05-26 10:57:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of Substance P significantly increases the total number of ipsilateral-cheek directed scratching bouts. A) \u003c/strong\u003eSubstance P KO mice had significantly more ipsilateral cheek directed scratching bouts as compared to WT controls following intradermal histamine (100 µg/20 µL) injection. \u003cstrong\u003eB)\u003c/strong\u003eThere 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. \u003cstrong\u003eC)\u003c/strong\u003e 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 \u003cstrong\u003eD)\u003c/strong\u003ePharmacological inhibition of TacR1 with L-733,060 (1 mg/kg in 100 µL I.V.) did not significantly change the number of dorsal nape-directed scratching bouts following intradermal histamine (100 µg/20 µL) injections. \u003cstrong\u003eE)\u003c/strong\u003e BNP-KO mice exhibited significantly fewer ipsilateral cheek-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection. \u003cstrong\u003eF)\u003c/strong\u003e BNP-KO mice exhibited significantly fewer dorsal nape-directed scratching bouts following intradermal histamine (100 µg/20 µL) injection. \u003cstrong\u003eG) \u003c/strong\u003eIntraperitoneal 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. \u003cstrong\u003eH)\u003c/strong\u003e 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. Data is presented as Mean ± Standard Deviation. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 as determined by a 2-tailed Student’s t-test. Each data point represents 1 biological replicate.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/1dae962614939cc3a55fe1de.jpg"},{"id":83446180,"identity":"e562c15c-6b74-4107-b64a-8fb940ab0975","added_by":"auto","created_at":"2025-05-26 11:21:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecific activation of HRH3 with immethridine induced significantly fewer scratching bouts and significantly higher wiping bouts. A)\u003c/strong\u003eSchematic demonstrating which histamine receptors are activated by the agonists Histamine, 2-pyridylethylamine, or immethridine. \u003cstrong\u003eB)\u003c/strong\u003e immethridine (100 µg/ 20 µL) induces significantly fewer scratching bouts than histamine (100 µg/ 20 µL) or 2-Pyridylethylamine in the dorsal nape. \u003cstrong\u003eC) \u003c/strong\u003eimmethridine (100 µg/ 20 µL) induces significantly fewer scratching bouts than histamine (100 µg/ 20 µL) or 2-Pyridylethylamine in the ipsilateral cheek. \u003cstrong\u003eD) \u003c/strong\u003eimmethridine (100 µg/ 20 µL) induces significantly more ipsilateral cheek directed wiping bouts than histamine (100 µg/ 20 µL). Data is presented as Mean ± Standard Deviation, *p \u0026lt; 0.05, ***p \u0026lt; 0.001 as determined by a 1-way ANOVA with a \u003cem\u003eposthoc \u003c/em\u003eHolm – Šídák test for multiple comparisons. Schematic in panel \u003cstrong\u003eA\u003c/strong\u003e was made in BioRender.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/0aaebaf56821da45e89f06d9.jpg"},{"id":83446033,"identity":"812ceffe-d67d-49ea-8ce1-3e8bb976ecee","added_by":"auto","created_at":"2025-05-26 11:13:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein and gene expression showing SST and SP overlap in the TG of mice and human. A) \u003c/strong\u003eRepresentative immunohistochemical images of SST and SP positive DRG neurons isolated from Tac1-cre::Ai9 mice that were stained for SST. \u003cstrong\u003eB)\u003c/strong\u003e Representative immunohistochemical images of SST and SP positive TG neurons isolated from Tac1-cre::Ai9 mice that were stained for SST. \u003cstrong\u003eC)\u003c/strong\u003e Quantification of neurons positive both SST \u0026amp; SP normalized to the total number of SST-positive neurons. \u003cstrong\u003eD)\u003c/strong\u003e Venn Diagram demonstrates the relative percentage of DRG neurons positive for SST-only, SP-only, \u0026amp; both SST and SP. \u003cstrong\u003eE)\u003c/strong\u003e Venn Diagram demonstrates the relative percentage of TG neurons positive for SST-only, SP-only, \u0026amp; both SST and SP. \u003cstrong\u003eK) \u003c/strong\u003eRepresentative ISH images of SST and SP positive human-derived DRG neurons. \u003cstrong\u003eL)\u003c/strong\u003eRepresentative ISH images of SST and SP positive human TG neurons. \u003cstrong\u003eM)\u003c/strong\u003eQuantification of human DRG and TG neurons positive for SST, SP, or SST and SP normalized to the number of SST-positive neurons. \u003cstrong\u003eN)\u003c/strong\u003e Venn diagram demonstrating the relative percentage of human DRG neurons positive or SST, SP, or Both. \u003cstrong\u003eO)\u003c/strong\u003e Venn diagram demonstrates the relative percentage of human TG neurons positive for SST, SP, or both. Data is shown as Mean ± Standard Deviation, **p \u0026lt; 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) or N = 2 (human) samples.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/e7899a7ed4659676cb93aadc.jpg"},{"id":83445297,"identity":"2e2b860c-ca43-4570-a2ca-c7642c97b1b1","added_by":"auto","created_at":"2025-05-26 10:57:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRetrograde tracing shows neurons that express both SST and SP in the TG of mice that innervate the skin. A) \u003c/strong\u003eRepresentative \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH) images of SST and SP positive DRG neurons isolated from Wheat Germ Agglutinin injected (1mg/mL in 50 µL) C57Bl6/J mice that were stained for SST and SP. \u003cstrong\u003eB)\u003c/strong\u003e Representative immunohistochemical images of SST and SP positive TG neurons isolated from Wheat Germ Agglutinin injected (1mg/mL in 50 µL) C57Bl6/J mice that were stained for SST and SP. \u003cstrong\u003eC)\u003c/strong\u003eQuantification of neurons positive both SST \u0026amp; SP normalized to the total number of SST-positive neurons. \u003cstrong\u003eD)\u003c/strong\u003e Venn Diagram demonstrating the relative percentage of DRG neurons positive for WGA-only, SST-only, SP-only, \u0026amp; WGA, SST, and SP. \u003cstrong\u003eE)\u003c/strong\u003e Venn Diagram demonstrating the relative percentage of TG neurons positive for WGA-only, SST-only, SP-only, \u0026amp; WGA, SST, and SP. Data is shown as Mean ± Standard Deviation, **p \u0026lt; 0.01 as determined by a 2-tailed paired Student’s t-test. Arrows identify neurons positive for both SST and SP. Scale bar = 50 µm for all images. Similar results were seen in at N = 5 (mice) samples.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/e387ceca2f93de63d65c44a3.jpg"},{"id":83445295,"identity":"e8b3a8f7-963a-4ebf-be70-b422c75c63c4","added_by":"auto","created_at":"2025-05-26 10:57:35","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":107700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSkin-innervating TG sensory neurons have larger populations of neurons positive for HRH1, SST, and SP or HRH3, SST, and SP as compared to skin-innervating DRG sensory neurons. A) \u003c/strong\u003eRepresentative 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. \u003cstrong\u003eB) \u003c/strong\u003eRepresentative 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. \u003cstrong\u003eC) \u003c/strong\u003eRepresentative 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. \u003cstrong\u003eD) \u003c/strong\u003eRepresentative 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. \u003cstrong\u003eE)\u003c/strong\u003e The Venn diagram showing the sizes of the HRH1, SP, and SST populations normalized to WGA-350-positive DRG neurons. \u003cstrong\u003eF)\u003c/strong\u003e The Venn diagram showing the sizes of the HRH1, SP, and SST populations normalized to WGA-350-positive TG neurons. \u003cstrong\u003eG)\u003c/strong\u003e The Venn diagram showing the sizes of the HRH3, SP, and SST populations normalized to WGA-350-positive DRG neurons. \u003cstrong\u003eH)\u003c/strong\u003e The Venn diagram shows the sizes of the HRH1, SP, and SST populations normalized to WGA-350-positive 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.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/48ad64801090e02b2e208f4e.jpg"},{"id":83446034,"identity":"0becfe90-5f01-4a25-b03a-9689a150cc84","added_by":"auto","created_at":"2025-05-26 11:13:35","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":98789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman DRG and TG recapitulate the changes in DRG and TG sensory neuron populations of HRH1 or HRH3, SST, and SP. A)\u003c/strong\u003eRepresentative FISH images of Human DRG neurons stained for HRH1, SP, and SST. \u003cstrong\u003eB)\u003c/strong\u003e Representative FISH images of Human TG neurons stained for HRH1, SP, and SST. \u003cstrong\u003eC)\u003c/strong\u003eRepresentative FISH images of Human DRG neurons stained for HRH3, SP, and SST. \u003cstrong\u003eD)\u003c/strong\u003e Representative FISH images of Human DRG neurons stained for HRH3, SP, and SST. \u003cstrong\u003eE)\u003c/strong\u003eVenn diagram showing the relative sizes of the HRH1, SP, and SST populations normalized to the number of counted DRG neurons. \u003cstrong\u003eF)\u003c/strong\u003e Venn diagram showing the relative sizes of the HRH1, SP, and SST populations normalized to the total number of counted TG neurons. \u003cstrong\u003eG)\u003c/strong\u003e Venn diagram showing the relative sizes of the HRH3, SP, and SST populations normalized to the total number of counted DRG neurons. \u003cstrong\u003eH)\u003c/strong\u003e The Venn diagram showing the sizes of the HRH3, SP, and SST populations normalized to the total number of counted 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 or TG human samples. Arrows identify neurons positive for all three genes of interest.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/c402a651f09f5db908f2cde2.jpg"},{"id":83445311,"identity":"af42b825-36ae-4700-bb98-5ff754025379","added_by":"auto","created_at":"2025-05-26 10:57:35","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":60590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of histamine-evoked itch in the dorsal nape and cheek. \u003c/strong\u003eInterstitial histamine activates HRH1 and/or HRH3 on DRG and TG sensory neurons. The majority of HRH1 and HRH3 expressions are limited to the SST population of itch mediating neurons in the DRG. In the TG, HRH1 and HRH3 are expressed on the SST-positive itch TG neurons and expressed in the SST-positive/SP-positive population of TG neurons that are polymodal for itch and pain signaling. The activation of these SST-positive/SP-positive neurons result in modulation of TG-mediated itch signals. Schematic was made in BioRender.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/5c78f86ebfc3c6206778c509.jpg"},{"id":83446795,"identity":"0a79f5ec-68f3-4ed8-bcbe-12bc12ad0cf5","added_by":"auto","created_at":"2025-05-26 11:29:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1841735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6466187/v1/786c8725-91e4-4ee9-8a05-dc20abcae427.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The Molecular and Cellular Basis of Regional Itch Differences","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSomatosensory 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 \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and humans\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. 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\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, but whether that induces a differential behavioral response or differences in itch circuitry in mice is unknown.\u003c/p\u003e \u003cp\u003eIn humans, the severity of itch is perceived differentially across body regions, with the face and torso\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e 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\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This difference is consistent across common pruritogens\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, including histamine\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Notably, facial injection of pruritogens and algogens activates an overlapping population of second-order neurons found in the spinal trigeminal nucleus\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In contrast, itch and pain are processed in the DRG sensory neurons and activate separate populations of second-order neurons\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe distinct neural processing of itch in the TG versus the DRG suggests the involvement of specific neuropeptides and signaling pathway involved in transmitting itch signals from the face versus the body. Itch and pain sensations are mediated through specific neuropeptides. Substance P (SP) mediates pain sensations\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Meanwhile, itch sensations are mediated by somatostatin (SST)-expressing neurons, which are \u0026gt;\u0026thinsp;99% co-localized with brain natriuretic peptide (BNP)-expressing neurons\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Activation of these first-order neurons triggers the release of these neuropeptides, which then activate second-order neurons in the dorsal horn of the spinal cord to further process these pain and itch sensations\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe significant difference in itch processing between the face (cheek) and torso (neck) reveals a substantial knowledge gap. To address this, we employed a multifaceted approach combining mouse genetics and behavioral, pharmacological, cellular, and molecular techniques. Following intradermal histamine injections into the dorsal nape or cheek, we investigated itch behavior in C57Bl6/J (inbred) and CD-1 (outbred) mice. Additionally, we examined histamine-induced itch behavior in mice lacking BNP or SP as well as in mice treated with antagonists for their respective receptors (NPR1\u003csup\u003e24\u003c/sup\u003e and TACR1). We also assessed nape and cheek skin innervation density by genetically marking somatostatin-expressing neurons with tdTomato. To identify differences in neuronal populations, we used tracing techniques followed by immunohistochemistry and \u003cem\u003ein-situ\u003c/em\u003e hybridization to analyze histamine receptors H1 and H3, BNP/SST, and SP in human and mouse DRG and TG. In summary, these experiments aim to elucidate the mechanisms underlying the differences in itch processing between the face and body.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRegional scratching response between the face and neck\u003c/h2\u003e \u003cp\u003eWe initially performed a literature review of papers published between January 2009 and June 2023 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,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\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), chloroquine (*p\u0026thinsp;=\u0026thinsp;0.0008), and SLIGRL-NH2, PAR-2 specific agonists (*p\u0026thinsp;=\u0026thinsp;0.0006) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This data was further confirmed when we combined all scratching bouts/minute values for all pruritogens (*p\u0026thinsp;=\u0026thinsp;0.0073) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo 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\u0026thinsp;=\u0026thinsp;0.0040) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) 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\u0026thinsp;=\u0026thinsp;0.2947) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and no significant differences were seen in the average bout duration (*p\u0026thinsp;=\u0026thinsp;0.2540) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The time course of histamine-induced scratching bouts differed significantly between the dorsal nape and cheek (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Ipsilateral cheek-directed scratching bouts peaked within the first 5 minutes (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) and the number of dorsal nape-directed scratching bouts peaked at 15 minutes (*p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-K)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Our results showed a similar pattern: fewer ipsilateral cheek-directed scratching bouts (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001); however, we found significantly shorter scratching times than dorsal nape-directed scratching bouts (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH \u0026amp;I). Unlike with C57BL/6J mice, bout duration significantly differed between regions in the CD-1 mice (*p\u0026thinsp;=\u0026thinsp;0.0380) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). The time course of histamine-induced scratching bouts differed between regions, with cheek-directed scratching peaking within 5 minutes, and significant differences were observed at 15 (*p\u0026thinsp;=\u0026thinsp;0.0002), 20 (*p\u0026thinsp;=\u0026thinsp;0.0477) and 30 minutes (*p\u0026thinsp;=\u0026thinsp;0.0009) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eOverall, our data suggests that regional differences in itch are conserved across outbred and inbred mouse lines, with the cheek being less responsive to histamine-induced itch compared to the dorsal nape.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRegional itch differences are not explained by innervation densities\u003c/em\u003e \u003c/p\u003e \u003cp\u003eGiven 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 TG\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and the dorsal nape dermatomes innervated by the DRG\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. 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 responsible for mediating itch signals induced by histamine\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. 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\u0026thinsp;=\u0026thinsp;0.0158) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC - E). 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\u0026thinsp;=\u0026thinsp;0.0029) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). This increase is likely to be responsible for increased SST-positive afferent density between the dorsal nape and the cheek. These findings also establish that the higher innervation density of itch-sensing neurons in the cheek does not correlate with the reduced scratching response observed in this region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eDistinct role of SP in modulating itch response in the cheek and dorsal nape.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSince innervation density did not explain the regional differences in itch response, we hypothesized that pain-associated neuropeptides, specifically Substance P (SP)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, might modulate ipsilateral cheek-directed scratching behavior. Based on previous observations where we found the dorsal nape, all SST-expressing DRG sensory neurons are positive for BNP\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Further, these BNP-expressing DRG sensory neurons are not positive for Substance P (SP)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, a canonical pain-associated neuropeptide.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e While there is literature about the relative co-expression of these neuropeptides in DRG sensory neurons, there is no information about their co-expression in TG sensory neurons.\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We found that SP-KO mice exhibited significantly more ipsilateral cheek-directed scratching bouts than wild-type controls (*p\u0026thinsp;=\u0026thinsp;0.0007) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with no differences in dorsal nape-directed scratching bouts (*p\u0026thinsp;=\u0026thinsp;0.2390) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We used BNP-KO mice as a control, as these mice should lack an itch behavior phenotype, and demonstrated that the loss of BNP resulted in a significant decrease in the total number of dorsal nape (*p\u0026thinsp;=\u0026thinsp;0.0126) \u003cem\u003eand\u003c/em\u003e ipsilateral cheek-directed (*p\u0026thinsp;=\u0026thinsp;0.0338) scratching bouts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE \u0026amp; F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that our behavior results in SP-KO and BNP-KO mice, we pharmacologically inhibited the SP receptor (TacR1) with L-733,060\u003csup\u003e31,32\u003c/sup\u003e. We found that these mice exhibited significantly higher amounts of ipsilateral cheek-directed scratching bouts than vehicle-treated mice (*p\u0026thinsp;=\u0026thinsp;0.0198) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 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\u0026thinsp;=\u0026thinsp;0.3568) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Mice treated with the NPR1 antagonist JS-11\u003csup\u003e33\u003c/sup\u003e experienced significantly fewer scratching bouts in both the ipsilateral cheek (*p\u0026thinsp;=\u0026thinsp;0.0338) and the dorsal nape (*p\u0026thinsp;=\u0026thinsp;0.0396) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG \u0026amp; 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.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHistamine receptor-specific itch induction.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo 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, while HRH3 is expressed in SP-positive DRG sensory neurons involved in pain\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo determine which histamine receptor(s) are responsible for transducing histamine-induced itch, we injected 2-pyridylethylamine (HRH1-specific agonist)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and immethridine (HRH3-specific agonist)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e 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\u0026thinsp;=\u0026thinsp;0.5208, nape; *p\u0026thinsp;=\u0026thinsp;0.7708, cheek) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; C). We found that 2-Pyr induced significantly more cheek wipes as compared to histamine in addition to the cheek directed scratching (*p\u0026thinsp;=\u0026thinsp;0.0230) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Immethridine also induced more ipsilateral cheek wipes (*p\u0026thinsp;=\u0026thinsp;0.136) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), but fewer scratching bouts than histamine (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, nape; *p\u0026thinsp;=\u0026thinsp;0.0264, cheek) in both regions. Immethridine induced fewer scratching bouts than 2-pyridylethylamine in the nape (*p\u0026thinsp;=\u0026thinsp;0.0002, nape), but not the cheek (*p\u0026thinsp;=\u0026thinsp;0.2373). Our results suggest that HRH1 is the primary histamine receptor mediating itch in dorsal nape-innervating DRG sensory neurons, while HRH3 may play a role in pain-behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRegional variations in sensory neuron populations underlying itch and pain\u003c/h3\u003e\n\u003cp\u003eOur previous findings on regional differences in itch and SST/SP innervation led us to investigate the populations of SST-, SP-, 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 behavior 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\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, 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\u0026thinsp;=\u0026thinsp;0.0012) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026ndash; D). We further validated this in human using \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing wheat germ agglutinin (WGA-350) as a neuronal tracer, used here to identify skin-innervating sensory neurons, we also 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\u0026thinsp;=\u0026thinsp;0.0051) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Next, we investigated the expression of HRH1 and HRH3 in skin-innervating sensory neurons in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our results showed an increase in the percentage of TG sensory neurons expressing HRH1, SST, and SP, as well as HRH3, SST, and SP, compared to the DRG. We further corroborated these findings in human TG versus DRG regional-specific differences in human sensory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study reveals significant regional differences in itch response between the cheek and dorsal nape (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These differences are not attributed to variations in innervation density (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but to distinct populations of sensory neurons expressing specific neuropeptides and receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Here, we demonstrate that the difference in facial histaminergic itch processing is due to differences in the proportion of TG neurons that co-express Substance P with SST, HRH1, and HRH3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e)). Behaviorally, this results in the likely activation of HRH1, inducing pain responses in the cheek. Further, we demonstrate that HRH3 might transduce histamine activation into pain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Our histological results were also recapitulated in human DRG and TG samples (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), extending their translational relevance. Finally, our results indicate that the TG processes facial itches fundamentally differently by a mechanism similar to what is seen in the gate control model of itch and pain processing (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHere, we confirmed that the number of histamine-induced ipsilateral cheek-directed scratching bouts is significantly reduced as compared to the number of histamine-induced dorsal nape scratching bouts by systematically comparing the scratching bouts by using the C57Bl/6J mice and the same amount of histamine in the same injection volume for both the cheek and nape injections. These results confirm that the reduction in the total number of ipsilateral cheek-directed scratching bouts is due to a difference in the neurophysiology of the cheek skin as opposed to differences in injection volumes or dose (at least for histamine). Interestingly, outbred CD-1 mice, which are thought to be a better model for human research\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, had significantly fewer ipsilateral cheek-directed scratching bouts, corroborating the results from the inbred line. Furthermore, CD-1 mice have significantly shorter scratching bout durations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), implying that they also perceive histaminergic itch in the cheek as less intense than histaminergic itch in the dorsal nape. Validation of regional differences in histamine-induced itch between inbred and outbred lines suggests that the study findings are not limited to a specific mouse strain and likely translatable to humans.\u003c/p\u003e \u003cp\u003e One of the limitations of our study is that we specifically investigated the role of histamine in regional itch differences, despite literature suggesting that other itch mediators, such as chloroquine, serotonin, SLIGRL-NH2, IL31, and proteases, may also exhibit similar regional differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There are mostly three reasons for this focus: Firstly, histamine is a well-known endogenous mediator for itch. Further, its involvement in itch processing is a helpful model for understanding the neural mechanisms underlying regional differences itch sensitivity. Secondly, in sensory neurons, histamine's-induced itch primarily mediated by the histamine receptor 1 (HRH1). This receptor specificity allows for targeted investigation of histamine's role in regional itch differences, without the confounding effects of other itch mediators. Lastly, histamine allows for a more in-depth investigation of the neural mechanisms underlying regional itch differences, which can serve as a foundation for future studies on other itch mediators, and the clinical relevance of histamine in itch disorders makes it a prime target for therapeutic intervention at sensory neurons carrying message to the CNS.\u003c/p\u003e\n\u003ch3\u003eHistamine-induced behavior hints at TG-specific processing of itch behavior\u003c/h3\u003e\n\u003cp\u003eReports have indicated differences between the DRG and TG at the transcriptome level\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Yet, the overall assumption has been that itch and pain processing in the TG follows the same pathways as itch processing in the DRG. Electrophysiology work performed by Akiyama et al., \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e further hinted at a difference in pain and itch signaling when they reported that pruritogens and algogens, itch inducers and pain inducers, respectively, results in postsynaptic activation of an overlapping population of neurons in the spinal trigeminal (TG) nucleus.\u003c/p\u003e \u003cp\u003eThere are currently two main theories about how the nervous system processes itch and pain. The first is a modified label-line theory, which posits that itch and pain are processed by two parallel pathways by the peripheral nervous system and the spinal cord dependent on the input simulus\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Overall, the label line theory is correct for the DRG; SST-positive/BNP-positive neurons primarily process itch, and pain is mediated mainly by SP-expressing DRG neurons\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Further supporting this theory is the finding that activation of the MrgprA3-expressing DRG neurons by capsaicin in TRPV1-KO::MrgprA3\u003csup\u003eROSA\u0026thinsp;\u0026minus;\u0026thinsp;TRPV1\u003c/sup\u003e mice, wherein TRPV1 is \u003cem\u003eonly in MrgprA3-expressing neurons\u003c/em\u003e, results in itch \u0026ndash; \u003cem\u003enot\u003c/em\u003e pain\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The second theory of itch and pain processing is the Gate Control theory, which states that separate pain and itch-responding DRG neurons activate the same set of secondary neurons located in the dorsal horn of the spinal cord. This theory was founded by discovery of dynorphin-expressing spinal interneurons activated following TRPV1 or TRPM8 neurons activation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Activation of these dynorphin-expressing interneurons triggers the release of dynorphin, which then suppresses activation of gastrin-related peptide receptor (GRPR)-expressing spinal interneurons, thus preventing the transmission of itch signals\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Current research has determined that these GRPR-expressing spinal interneurons are where itch signals converge\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e before these interneurons transmit itch signals to the parabrachial nucleus\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This theory is further supported by the finding that SST-dependent activation of SSTR2-subpopulation inhibits pain responses \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Both of these theories on itch processing have been assumed to be the \u003cem\u003ede facto\u003c/em\u003e pathways by which the TG processes itch behavior despite the lack of evidence, either for or against, the presence of these pathways in the TG nucleus.\u003c/p\u003e \u003cp\u003eOur results suggest that \u003cem\u003ethe TG itself uses gate control to process facial itch signals\u003c/em\u003e despite the unclear presence of these pathways in the spinal trigeminal nucleus\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Our mouse data demonstrate that TG have a significantly increased percentage of SST-expressing neurons expressing Substance P compared to the DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). HRH1, which transduces histamine receptor activation into itch, has significant changes in downstream neuropeptide populations (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In the DRG, HRH1 is predominantly co-expressed with SST, with few neurons being positive for HRH1 and Substance P; however, in the TG, this proportion of HRH1-positive and Substance P-positive neurons roughly triples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This change in HRH1 and Substance P expression also results in a change in behavioral outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). HRH3 underwent similar changes in the proportion of neurons positive for Substance P in the TG compared to the DRG (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Interestingly, immethridine, an HRH3-specific agonist\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, did not induce itch but significantly more ipsilateral cheek-directed wipes than histamine, indicating that histaminergic itch is both receptor-dependent and neuropeptide-dependent. These findings provide a basis for examining how the gate control theory of pain and itch processing may be relevant in this context. The gate control theory of pain and itch processing has evidence that there might be two subtypes: the leaky gate subtype suggesting itch and weak pain signals activate the same pathway\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and an intensity coding subtype suggests itch signal transmission is dependent on a frequency or burst of stimulation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. While we could not test this hypothesis here, but our results, and those reported in Akiyama et al\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, alluding to an intensity coding in the TG. Another avenue to explore would be to determine if the histamine-triggered SP-release can activate a previously identified monosynaptic facial pain circuit\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e as these neurons express the receptor for SP\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Activation of this pathway by histamine would also likely account for the decrease in itch behavior and increase in pain following intradermal injection of histamine into the cheek.\u003c/p\u003e \u003cp\u003eIn summary, we identified distinct sensory neuron subpopulations, highlighting the importance of considering regional differences in itch response when designing treatment strategies. Overall, our study has significant implications for the understanding and treatment of itch disorders by developing therapies targeting HRH1 and HRH3 receptors that may provide new treatment options for itch disorders.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eHistamine 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-Pyrdylethylamine (Catalog # 2478), and immethridine (Catalog # 2315) were ordered from Tocris. JS-11 (1-cyclohexyl-3-(cyclopropylmethyl)-N-((3-methylisoxazol-5-yl)methyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide) was custom synthesized by ChemBridge.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eAll mice were housed at NC State University and all experiments were IACUC approved (protocols 22-167-B, 19 -047-B, and 16-038-B). Mice were given food and water \u003cem\u003ead libitum\u003c/em\u003e and kept on a 12-hour light dark cycle (6am -6pm EST).\u003c/p\u003e \u003cp\u003eAll 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-\u003cem\u003eTac1\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Bbm\u003c/em\u003e\u003c/sup\u003e/J (Substance P (SP) KO) (Strain # 004103)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eSst\u003c/em\u003e\u003csup\u003e\u003cem\u003etm2.1(cre)Zjh\u003c/em\u003e\u003c/sup\u003e/J (SST-cre) (Strain # 013044)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and B6.Cg-\u003cem\u003eGt(ROSA)26Sor\u003c/em\u003e\u003csup\u003e\u003cem\u003etm9(CAG0tdTomato)Hze\u003c/em\u003e\u003c/sup\u003e/J (Ai9) (Strain # 007909)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. BNP KO mice were a generous gift from Dr. Mark Hoon (NIH)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Crl:CD1(ICR) (CD-1) mice were ordered from Charles River laboratories (Strain # 022). SST-cre::Ai9 mice were generated by crossing the SST-cre and Ai9 lines.\u003c/p\u003e\n\u003ch3\u003eItch Behavior\u003c/h3\u003e\n\u003cp\u003eHistamine injection: All mice received intradermal injections of 100 \u0026micro;g/20 \u0026micro;L histamine in sterile 1X phosphate buffered saline (1X PBS). This concentration and volume were used for all intradermal injections. Immethridine and 2-Pyridylethylamine were intradermally injected with the same 100 \u0026micro;g/20 \u0026micro;L concentration. Mice were injected in either Dorsal Nape\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e or Cheek\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. L-733,060 was dissolved in sterile 1X PBS and 50 \u0026micro;L of a 1mg/kg solution was administered intravenously 30 minutes prior to histamine injection\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. 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 \u0026micro;g/50 \u0026micro;L of JS-11 was administered intraperitoneally 30 minutes prior to histamine injection\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe same mice were used for both the Cheek and Nape tests. For drug administration injections, mice were randomly assigned to receive Vehicle, L733-060, orJS-11. Mice were then randomized to the injection site. After a 10-day washout period, mice were treated with vehicle were then treated with JS-11 or L-733,060 and mice treated with JS-11 or L-733,060 were treated with vehicle.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eFor Tuj antibody staining, DRG and TG were isolated from SST-cre::Ai9 mice, fixed in 4% PFA for 24 hours at 4\u0026deg;C, cryoprotected using a 30% sucrose solution incubation (~\u0026thinsp;72 hrs at 4\u0026deg;C), and then frozen in OTC media. Sections were cut at 14 \u0026micro;m and placed in 4% PFA for 15 minutes at room temperature. Slides were then blocked with 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 slides, which were then incubated overnight at 4\u0026deg;C. After 16\u0026ndash;24 hrs, the slides were washed in ice cold 1X PBS trice and then incubated for 45 minutes at room temperature with Alexa Fluor 488 conjugated goat anti-rabbit IgG (Thermofisher, catalog # A-11008). Samples were images on a Leica DM500B epifluorescent microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSkin Innervation Tracing\u003c/h2\u003e \u003cp\u003eMice 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 neurons\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. WGA-350 was injected intradermally into both cheeks and the dorsal nape. Both TG and C3-T2 DRG were isolated 24 hours after injection\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. TG and DRG were then placed in OCT media and quickly frozen on dry ice and were stored at -80\u0026deg;C for up to 3 months. Cheek injections were bilateral to obtain the greatest potential number of TG sensory neurons that innervate the skin.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFluorescent\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eIn Situ\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHybridization\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIn 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 hours after receiving 50 \u0026micro;L of a 1mg/mL solution of Alexa Flour-conjugated Wheat Germ Agglutinin (WGA-350) and frozen in OTC media at -80\u0026deg;C within 30 minutes. These sections were cut at thickness of 10 \u0026micro;m and placed onto Superfrost Gold Plus Slides (Fisher, cat. # 15-188-48). After sectioning, sections were air dried at -20\u0026deg;C for 2 hours. After this, slides were placed in 4% paraformaldehyde (4% PFA) made with DEPC water (Thermofisher, Catalog # 750023) at 4\u0026deg;C for 15 minutes. Slides were then washed twice in 1X PBS made with DEPC water. Following this wash step, slides were dehydrated at room temperature in 50% ethanol for 5 minutes, 70% ethanol for 5 minutes, and two washes in 100% ethanol for 5 minutes. The 50% ethanol and 70% ethanol solutions were made with DEPC water. The slides were then kept at -20\u0026deg;C in 100% ethanol overnight. Ethanol was purchased from Millipore Sigma (Catalog # 459836-2L).\u003c/p\u003e \u003cp\u003eThe following day, the slides were removed from 100% ethanol and allowed to air-dry at room temperature for 5 minutes. 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 minutes. After drying, roughly 5 drops of RNAscope hydrogen peroxide was added to fill the area within the hydrophobic barrier on the slide and incubated for 10 minutes at room temperature. The slides were then washed in DEPC treated water. After this wash, roughly 5 drops of RNAscope Protease III was added to fill the area within the hydrophobic barrier and incubated for 30 minutes at room temperature. The slides were then washed twice in 1X PBS made with DEPC water. Sections were hybridized and on the same day; therefore, sodium citrate was not used.\u003c/p\u003e \u003cp\u003eFor hybridization, probes 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), and mouse Hrh3 (Catalog # 428481). 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 ~\u0026thinsp;6 drops of the appropriate probe mixture and then incubated for 2 hours at 40\u0026deg;C in a HybEZ oven. After this incubation, slides were washed in 1X wash buffer twice for two minutes (each wash). For the first two amplification steps, ~\u0026thinsp;6 drops of RNAscope Multiplex FL v2 Amp1 or Amp2 was added to the area within the hydrophobic barrier and incubated for 30 minutes at 40\u0026deg;C. For the third hybridization step, RNAscope Multiplex v2 Amp3 was added to the area within the hydrophobic barrier and incubated for 15 minutes at 40\u0026deg;C. After each hybridization step slides were washed twice in 1X was buffer for 2 minutes at room temperature.\u003c/p\u003e \u003cp\u003eTo 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 minutes at 40\u0026deg;C. Slides were then washed twice for 2 minutes in 1X wash buffer at room temperature. To the area in the hydrophobic barrier 200 \u0026micro;L 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\u0026deg;C for 30 minutes. All dyes were diluted 1:1000. After incubation, slides were washed twice in 1X wash buffer for 2 minutes at room temperature. Finally, roughly 6 drops of RNAscope Multiplex FL v2 HRP blocker was added to the area within the hydrophobic barrier and incubated at 15 minutes at 40\u0026deg;C. After this incubation, the slides were washed twice in 1X wash buffer for 2 minutes at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting and Quantification:\u003c/h2\u003e \u003cp\u003eImages 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e cell populations were normalized to the total number of cells counted. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the percentage of SST-expressing neurons was normalized to the total number of TUJ positive neurons counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eData was analyzed in Graphpad Prism. Paired 2-tailed t-tests were performed to compare histamine-induced scratching in the cheek versus dorsal nape. Region-based data was analyzed with a 2-way ANOVA with a \u003cem\u003epost hoc\u003c/em\u003e Holm \u0026ndash; Š\u0026iacute;d\u0026aacute;k test for multiple comparisons. Other data was compared with a 1-way ANOVA with a \u003cem\u003epost hoc\u003c/em\u003e Holm \u0026ndash; Š\u0026iacute;d\u0026aacute;k 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\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGEMENTS\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Mark Hoon for critically reviewing the manuscript and providing excellent feedback. This work was supported by NIH grant R01AR077692, intramural NCSU fundings and gift provided by Dr. John M. Davis.\u003c/p\u003e\n\u003cp\u003eCONFLICTS OF INTEREST\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoswami SC et al (2014) Molecular signatures of mouse TRPV1-lineage neurons revealed by RNA-Seq transcriptome analysis. 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Neurotoxicology 30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neuro.2009.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.neuro.2009.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evon Buchholtz LJ, Lam RM, Emrick JJ, Chesler AT, Ryba N (2020) J. P. Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning. Pain 161:2212\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6466187/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6466187/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eItch 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. Here, we investigate the neural basis of regional itch differences, focusing on the role of neuropeptides and histamine receptors expression. Using a combination of behavioral, pharmacological, genetic, histological, and molecular approaches, we identified distinct regional differences in itch response between the cheek and dorsal nape (neck). Our findings reveal that Substance P modulates itch in the cheek, while histamine receptor 1 (HRH1) is the primary receptor mediating itch but not pain in the cheek. We also discovered regional differences in sensory neuron subpopulations, with increased co-expression of HRH1 and neuropeptides in trigeminal ganglion neurons. In brief, we provide new insights into the molecular and cellular mechanisms underlying regional itch differences, highlighting the importance of considering regional variations in itch response to develop effective treatments.\u003c/p\u003e","manuscriptTitle":"The Molecular and Cellular Basis of Regional Itch Differences","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-26 10:57:30","doi":"10.21203/rs.3.rs-6466187/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d3c6bc2b-e57e-45ba-9112-753bbc091e8e","owner":[],"postedDate":"May 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47731926,"name":"Biological sciences/Neuroscience/Sensory processing"},{"id":47731927,"name":"Biological sciences/Neuroscience/Somatosensory system/Pain"}],"tags":[],"updatedAt":"2025-05-26T10:57:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-26 10:57:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6466187","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6466187","identity":"rs-6466187","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}