Functional roles of neuromedin B and gastrin-releasing peptide in regulating itch and pain in the spinal cord of non-human primates

Abstract

Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.

1. Introduction

Of all nociceptive somatosensory systems, acute itch/pruritus is an essential alert system that eliminates noxious components by scratching responses. However, intractable itch associated with several skin or systemic diseases (e.g., atopic dermatitis, dry skin, diabetes mellitus,and cholestatic liver disease) significantly affects the quality of life and causes huge economic losses globally [1,2]. Several lines of evidence reveal critical molecules that contribute to the transmission of itch from the periphery to the central nervous system [3–5]. The intrathecal (i.t.) administration of gastrin-releasing peptide (GRP), a mammalian bombesin-related peptide, elicits robust itch-related scratching behavior in rodents and primates [6,7]. Moreover, the ablation of GRP receptor (GRPR)⁺ neurons in the spinal dorsal horn (SDH) markedly reduces scratching behavior induced by different types of pruritic stimuli, without affecting the pain threshold in rodents [8,9]. The enhancement of GRP-GRPR activity based on the upregulation of each molecule and the sensitization of GRPR⁺ neurons may not only underlie acute but also chronic itch [10,11].

Neuromedin B (NMB) is a decapeptide that bears close homology with bombesin-related peptide, and it mediates several physiological functions such as smooth muscle contraction, hormone secretion, and cell growth [12,13]. In contrast to GRP, NMB may be involved in both itch and pain signaling. Initially, intracerebroventricular administration of NMB elicits itch-related scratching behavior [14], supported by other rodent studies using i.t. or an intracerebroventricular delivery route [15–17]. However, the degree of NMB-induced scratching is considerably less than that of GRP. Conversely, the intraplantar administration of NMB elicits nociceptive hypersensitivity and the ablation of NMB receptor (NMBR)⁺ neurons results in thermal hypoalgesia [18]. NMBR and GRPR, which have close structural homology, are predominantly expressed in separate subpopulations of spinal neurons, and the NMB-NMBR system may function upstream of the GRP-GRPR system in rodents [15,19]. Despite accumulating evidence in rodents, there is no evidence regarding the functional role of spinal NMB-NMBR system for regulating itch and pain in primates.

A substantial neurochemical gap between rodents and primates [20–22] warrants confirming the functional significance of key molecules contributing to sensory processing in primates. The pharmacological effects of opioid peptides (i.e., μ-opioid peptides and nociceptin/orphanin FQ) on pain and itch-related behaviors in non-human primates (NHPs) differ from those in rodents [23–25]. The i.t. administration of μ-opioid receptor agonists, such as morphine, produces itch sensation in patients and elicits robust scratching behavior in NHPs [6,26]; however, the degree of scratching in rodents is much less than that in NHPs [16,27,28]. Unlike opioids, the behavioral effects of i.t. GRP in rodents are similar to those in NHPs [6,29], thereby indicating the functional significance of GRP-GRPR system in rodents can be translated to NHPs. NMBR is distributed in the central nervous system of primates [30], thus functional evidence for the NMB-NMBR system in NHPs is needed to understand the mechanisms in the spinal transmission of itch. We aimed to compare the expression patterns of NMB-NMBR and GRP-GRPR systems and the behavioral effects of i.t. NMB with GRP on itch or pain sensation in NHPs.

2. Materials and methods

2.1. Animals

We used six adult rhesus monkeys (Macaca mulatta), comprising four males and two females, with a body weight of 6–11 kg and aged 10–16 years. The animals were housed at an indoor facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (Frederick, MD, USA). They were individually placed in cages with a floor space of 6–12 square feet, 2.7–5.4-feet high ceiling, and an environmentally controlled room (21–25 °C, 40–60% relative humidity) with a 12-h light/dark cycle (lights on: 6:30–18:30). They were provided with water, monkey chow (LabDiet St. Louis, MO, USA), and fresh fruit ad libitum. We provided them with primate enrichment devices and treats daily. They were not subjected to any experiment 1 month before initiating the study. The animals were assigned to each experiment based on the tasks they were trained to perform. All experiments followed a within-subject design (i.e., each group of animals served as its own control and all dosing conditions were randomized by a counterbalanced design). All experiments were conducted during late mornings of weekdays until the completion of time courses or testing sessions. All animal care and experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health (Bethesda, MD, USA), and were approved by the Institutional Animal Care and Use Committee of Wake Forest University (Winston-Salem, NC, USA). The present study was reported in accordance with the Animal Research: Reporting of In Vivo Experiments [31].

2.2. Tissue collection

Researchers have previously described detailed procedures for tissue collection [32]. Briefly, the animals were deeply anesthetized with sodium pentobarbital (Sigma-Aldrich, St. Louis, MO, USA) and the vascular system was flushed with saline (Baxter, Deerfield, IL, USA). The spinal column was dissected to expose the spinal cord and the attached dorsal root ganglia (DRG). For reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), the spinal dorsal horn (SDH) and DRG tissues from the lumbar segment 4 (L4) were flash frozen in dry ice. For in situ hybridization and immunohistochemistry, one block of L4 spinal cord tissue was fixed in 4% paraformaldehyde (Sigma-Aldrich), cryopreserved in 30% sucrose (Sigma-Aldrich) prepared in phosphate buffered saline (PBS) (Sigma-Aldrich), and embedded in an optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). All tissues were stored at −80 °C until use.

2.3. RT-qPCR

We performed SDH and DRG tissue processing and the RT-qPCR protocol according to a previous study [32]. The mRNA expression levels of NMB, GRP, NMBR, and GRPR from five animals were quantified based on the comparative threshold cycle (C_T) method [33]. PCR amplification efficiency for each primer was determined by the slope of standard curve generated from tenfold serial dilutions (0.02–20 ng) of the cDNA mixture from subjects. The average C_T values were obtained from three replicates and normalized against that of β-actin (ACTB) using the following formula: 2^{−(C_T target gene−C_T ACTB)} (2^−ΔC_T).

2.4. Immunohistochemistry

Fixed frozen spinal cord tissues were sectioned at a thickness of 30 μm using a cryostat (Leica CM3050S; Leica Biosystems, Wetzlar, Germany) for free-floating immunostaining. The tissue sections were washed in PBS containing 0.3% Triton X-100 (Sigma-Aldrich) and treated with TrueBlack Lipofusin Autofluorescence Quencher (Biotium, Fremont, CA, USA) for 2 min. After washing with PBS, we blocked the tissues with PBS containing 3% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 1 h. The sections were incubated with a primary antibody against GRPR (rabbit polyclonal, 1:500 dilution; MC-831, MBL International, Woburn, MA, USA), NMBR (rabbit polyclonal, 1:500 dilution; ABR-004, Alomone Labs, Jerusalem, Israel), and NeuN (mouse monoclonal, 1:500 dilution; MAB377, MilliporeSigma, Burlington, MA, USA; markers for neuronal nuclei) at 4 °C overnight. The following day, we rinsed the sections in PBS before incubation with donkey anti-rabbit or anti-mouse IgG secondary antibody conjugated to AlexaFluor 594 or 488, respectively (1:500 dilution; Jackson ImmunoResearch Laboratories), at room temperature for 3 h. All antibodies were prepared in PBS containing 1% normal donkey serum. The tissue sections were rinsed in PBS, mounted on glass slides to air dry, and cover-slipped with Prolong Gold AntiFade mounting media (ThermoFisher Scientific, Waltham, MA, USA).

2.5. In situ hybridization

Fixed frozen spinal cord tissues were sectioned at a thickness of 10 μm using a cryostat (Leica) for detecting mRNA expression. We performed in situ hybridization according to the protocol of the RNAscope Multiplex Fluorescent v2 Assay (Advanced Cell Diagnostics, Newark, CA, USA). Briefly, the spinal cord sections were fixed in 4% paraformaldehyde, dehydrated in ethanol, and treated with H₂O₂, target retrieval reagent, and protease III. The tissues were hybridized with commercial probes for NMBR (# 821681, GRPR (# 821671), protein kinase C γ (PKCG; # 837971; marker for pain processing neurons), vesicular glutamate transporter 2 (VGLUT2; # 540961; marker for excitatory neurons), and vesicular GABA transporter (VGAT; # 428881; marker for inhibitory neurons). Moreover, we visualized the nuclei with the DAPI. The tissue sections were cover-slipped with Prolong Gold AntiFade mounting media, followed by the capture of confocal images using Olympus FV1200 confocal microscope (Olympus America, Center Valley, PA, USA). For quantifying the percentage of co-expression, we captured mosaic images with Z series by setting a fixed matrix that contained the entire spinal dorsal horn. To quantify the distribution of NMBR⁺ and GRPR⁺ cells, we identified laminae I-III versus IV-V using standard lamination criteria [34]. Cells containing at least three dots of punctate staining were counted with the software Image J, developed by the National Institute of Health (NIH, Bethesda, MD, USA).

2.6. Itch scratching responses

We recorded the scratching responses of six animals in their home cages to assess the itching sensation caused by test compounds [35]. Each 15-min recording session followed the i.t. administration of NMB, GRP, or a mixture of NMB or GRP with different antagonists. The number of scratches was the primary outcome. A scratch was defined as one brief (<1 s) scraping on the skin surface of other body parts using the forepaw or hind paw. The total number of scratches for each 15-min period was calculated by experimenters blinded to the dosing conditions.

2.7. Acute thermal nociception

We performed the warm water tail-withdrawal assay to measure nociceptive responses of five animals to thermal stimuli and the effects of test compounds [36,37]. The animals were seated in primate restraint chairs, followed by the immersion of lower parts of their shaved tails (~15 cm) in water maintained at 42 °C, 46 °C, or 50 °C. We used water at 42 °C or 46 °C as a normally non-noxious stimulus (i.e., no tail-withdrawal expected), whereas that at 50 °C was used as an acute noxious stimulus (i.e., 2–3 s tail-withdrawal latency); however, it did not cause thermal injury. The tail-withdrawal latency was the primary outcome. The animals were randomly assigned to the dosing condition. Experimenters unaware of the dosing conditions measured the tail-withdrawal latencies at each temperature, randomly using a computerized timer. They recorded a maximum duration of 20 s (the cut-off) if the animal did not withdraw its tail within 20 s. Test sessions commenced with the baseline measurements at each temperature. Subsequently, the tail-withdrawal latencies were measured at 15 min, 30 min, 45 min, and 60 min following the i.t. administration of the test compound.

2.8. Capsaicin-induced thermal allodynia

We evaluated the antiallodynic effects of the test compound using a 1 h pretreatment regimen (i.e. 1 h before capsaicin administration) [38,39] in five animals. Capsaicin (1.2 mg/mL × 0.3 mL) was topically administered via a bandage attached to the terminal 3–5 cm of the tail for 15 min. The allodynic response was manifested as reduced tail-withdrawal latency from a maximum value of 20 s to approximately 2–3 s in 46 °C water. The aforementioned allodynic effect peaked at 15 min following the removal of the capsaicin bandage, during which we measured the tail withdrawal latency in 46 °C water (i.e., to determine the antiallodynic effects of the test compound).

2.9. Drugs

NMB (Sigma-Aldrich), RC-3095 (Sigma-Aldrich), GRP (Tocris Bioscience, Minneapolis, MN, USA), and DAMGO ([D-Ala²,N-MePhe⁴, Gly-ol⁵]-enkephalin) (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in sterile water. PD168368 (Tocris) stock solution was prepared in a 1:1:8 ratio of dimethyl sulfoxide (ThermoFisher Scientific), Tween 80 (ThermoFisher Scientific) and sterile water, and diluted with sterile water to obtain the target working solution. The doses of these test ligands were based on previous NHP studies [6,35,40]. We administered the drugs intrathecally following a previously described drug delivery procedure [35]. Briefly, the animals were positioned in primate restraint chairs and anesthetized by intravenous propofol (2.5–4.0 mg/kg for bolus infusion and 0.3–0.4 mg/kg/min for continuous infusion; AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA). A spinal needle (22-gauge × 1.5; BD Biosciences, Franklin Lakes, NJ, USA) was inserted into the subarachnoid space between the L4/L5 lumbar vertebra. A 1-mL solution of compound was slowly infused through the spinal needle within 30 s. Animals recovered from anesthesia within 10 min after the termination of propofol infusion, and were returned to their home cages.

2.10. Statistical analyses

Data are presented as mean values ± SD. We performed comparisons for similar monkeys across all test sessions for one experiment. We conducted the paired t-test (Fig. 1) and two- or one-way repeated measures analysis of variance (ANOVA) (Figs. 2, 3, and 4) to compare the outcome measures (i.e., tail-withdrawal latency and the number of scratches). The criterion for significance was set at p-value < 0.05. We used GraphPad Prism version 9 software for statistical analyses. Behavioral data collection was performed under blinded conditions. No statistical power calculations were performed prior to the study. The sample size was based on our previous experience with this design [38,40].

Fig. 1.

A comparison of mRNA expression of GRP versus NMB, and GRPR versus NMBR, in the dorsal root ganglia and spinal dorsal horn of monkeys. mRNA levels were quantified using RT-qPCR and analyzed by the comparative C_T method. (A) PCR amplification efficiency for each primer (NMB, GRP, NMBR, GRPR, and ACTB) was determined by the slope of standard curve generated from tenfold serial dilutions (0.02–20 ng) of the cDNA mixture from subjects. (B-E) Relative mRNA expression levels in the dorsal root ganglia (B, C) and spinal dorsal horn (D, E). Data represent the mean ± SD (n = 5), with dots representing individual NHP. Data were analyzed by the paired t-test. *p < 0.05, significantly different between GRP and NMB (B, D) or GRPR and NMBR (C, E).

Fig. 2.

Effects of intrathecal NMB and GRP on itch scratching responses in monkeys. (A, B) Time courses of itch scratching responses elicited by varying doses of NMB and GRP. (C) Total number of scratches summed from the four time points displayed in A and B. (D, E) Time courses of itch scratching responses elicited by NMB (100 nmol) or GRP (10 nmol), in combination with the GRPR antagonist RC-3095 (100 nmol) or NMBR antagonist PD168368 (300 nmol). Data represent the mean ± SD (n = 6), with dots representing individual NHP. Data were analyzed by the two- (A, B, D, and E) or one-way (C) repeated measures ANOVA, followed by Dunnett’s multiple comparison test. *p < 0.05, significantly different from the vehicle condition.

Fig. 3.

Effects of intrathecal NMB and GRP on the thermal nociceptive threshold, compared with substance P in monkeys. (A-C) Time courses of tail-withdrawal latencies measured in 46 °C water following the intrathecal administration of varying doses of NMB (A), GRP (B), or substance P (C). Data represent the mean ± SD (n = 5), and were analyzed by the two-way repeated measures ANOVA, followed by Dunnett’s multiple comparison test *p < 0.05, significantly different from the vehicle condition.

Fig. 4.

Effects of intrathecal NMBR and GRPR antagonists on peripherally elicited pain, compared with DAMGO ([D-Ala²,N-MePhe⁴,Gly-ol⁵]-enkephalin) in monkeys. Topical capsaicin-induced allodynia was evaluated by tail-withdrawal assays in 46 °C water following the intrathecal administration of μ-opioid receptor agonist DAMGO (1 and 3 nmol), GRPR antagonist RC-3095 (100 and 300 nmol), or NMBR antagonist PD168368 (300 and 1000 nmol). Data represent the mean ± SD (n = 5), and were analyzed via a one-way repeated measures ANOVA, followed by Dunnett’s multiple comparison test. *p < 0.05, significantly different from the vehicle condition.

3. Results

3.1. Expression of NMB, GRP, and their receptors in the DRG and SDH

First, we investigated the gene expression levels of NMB, GRP, and their receptors in the DRG and SDH of NHPs by RT-qPCR. The slopes of the standard curves for each gene were almost identical, which indicated that the PCR amplification efficiencies were similar among same categories of molecules (Fig. 1A). In both the DRG and SDH, mRNA expression levels of NMB (relative to ACTB) were substantially greater than those of GRP (Fig. 1B, D), whereas the levels of NMBR were significantly lower than those of GRPR (Fig. 1C, E).

3.2. Effects of i.t. NMB and GRP on itch sensation

We conducted behavioral analyses to evaluate the effects of spinally-delivered NMB and GRP on itch sensation in NHPs. The i.t. administration of NMB transiently elicited scratching behavior in a dose-dependent manner (Fig. 2A, C). In contrast, the i.t. administration of GRP caused robust and prolonged scratching behavior (Fig 2B, C). NMB-induced scratching behavior was completely blocked by the NMBR antagonist (PD168368), but not the GRPR antagonist (RC-3095) (Fig. 2D). Conversely, GRP-induced scratching behavior was antagonized by RC-3095, but not PD168368 (Fig. 2E).

3.3. Effects of i.t. NMBR or GRPR ligands on pain sensation

Subsequently, we determined the impact of i.t. NMB and GRP on pain sensitivity in NHPs. Neither NMB nor GRP affected withdrawal latency to innoxious stimuli, whereas the i.t. administration of substance P reduced the latency to stimuli, thus displaying thermal allodynia (Fig. 3). Moreover, we assessed the anti-allodynic effects of PD168368 or RC-3095 on capsaicin-induced thermal allodynia. Unlike DAMGO, a μ-opioid receptor agonist with anti-allodynic effects, PD168368 and RC-3095 did not exert an effect on thermal allodynia over the behaviorally active dose range (Fig. 4).

3.4. Localization of NMBR in different subsets of neurons in the SDH

We characterized the distributions of NMBR and GRPR in the SDH. By immunohistochemistry with NeuN antibody that labels neuronal nuclei, protein expressions of NMBR and GRPR were located on the NeuN⁺ neurons mainly in the superficial laminae and much less in the deeper area (Fig. 5 A, B). By in situ hybridization using RNAscope, close to 90% of the NMBR⁺ neurons and GRPR⁺ neurons were distributed in laminae I-III and only about 10% were in laminae IV-V (Fig. 6A, D). Less than 20% of the NMBR was colocalized with GRPR (Fig. 6 A, E), and the majority of NMBR and GRPR did not colocalize with PKCG, a marker of pain-responsive neurons (Fig. 6B, C, F). Approximately 60% and 70% of the NMBR⁺ neurons and GRPR⁺ neurons, respectively, were VGLUT2⁺ excitatory neurons (Fig. 7A, B, E), whereas the minority of NMBR⁺ and GRPR⁺ neurons were VGAT⁺ inhibitory neurons in the SDH (Fig. 7C, D, F).

Fig. 5.

Expression of NMBR and GRPR protein in the spinal dorsal horn of monkeys. (A, B) Representative images of immunohistochemistry depict the expression of GRPR (left column) and NMBR (right column) in NeuN⁺ cells. (C) Higher magnification of the boxed areas in (B). Scale bar = 200 μm (A, B), and 50 μm (C).

Fig. 6.

Expression of NMBR and GRPR mRNA in the spinal dorsal horn of monkeys (A-C). Representative confocal images of in situ hybridization (RNAscope) depict the mRNA expression of NMBR in GRPR⁺ (A) or PKCG⁺ neurons (B) and the mRNA expression of GRPR in PKCG⁺ neurons (C). (D) Quantification of the distribution of NMBR⁺ and GRPR⁺ neurons in laminae I-III and IV-V of the spinal dorsal horn. (E, F) Bar graphs depict the percentage of co-expression. Data represent the mean ± SD (n = 3), with dots representing individual NHP. Scale bar = 20 μm.

Fig. 7.

Expression of VGLUT2 and VGAT mRNA in NMBR⁺ and GRPR⁺ neurons in the spinal dorsal horn of monkeys. (A-D) Representative confocal images of in situ hybridization (RNAscope) show the mRNA expression of VGLUT2 in NMBR⁺ (A) or GRPR⁺ (B) neurons and the mRNA expression of VGAT in NMBR⁺ (C) or GRPR⁺ (D) neurons. (E, F) Bar graphs show the percentage of co-expression. Data represent the mean ± SD (n = 3) with dots representing individual NHP. Scale bar = 20 μm.

4. Discussion

We provided three key findings to define the functional roles of the NMB-NMBR system in the SDH of NHPs. First, the i.t. administration of NMB elicited a milder scratching behavior with lower potency than GRP. Moreover, the activation of spinal NMB-NMBR or GRP-GRPR systems resulted in scratching responses via independent receptor mechanisms. Second, i.t. NMB and GRP did not induce thermal hypersensitivity. Neither did i.t. NMBR and GRPR antagonists modulate peripherally elicited thermal allodynia induced by capsaicin. Third, NMBR displayed lower expression in both itch- and pain-responsive neurons in the SDH, namely GRPR⁺ and PKCγ⁺, respectively. In other words, the spinal NMB-NMBR system plays a limited role in the neurotransmission of itch and pain in NHPs.

Exploring the functional roles of spinal NMB-NMBR and GRP-GRPR systems in regulating itch and pain in primates necessitates determining their expression patterns in the sensory neurons and SDH. Despite the functional significance of GRP in itch transmission [8,19,41,42], sources of GRP contributing to itch sensation are still controversial in rodents. GRP is produced in the interneurons located on the SDH [43–45], whereas a recent report demonstrated that functionally-important GRP is located on the sensory neurons, despite lower expression levels of GRP in the DRG than that in the SDH [46]. Nevertheless, expression patterns of GRP in NHPs are consistent with findings from rodent studies that GRP-expressing cells are principally located on the SDH, but not the DRG. However, NMB is produced by DRG neurons and contributes to nociceptive transmission in rodents [15,18,47]. The expression levels of NMB are greater than that of GRP in both the DRG and SDH of NHPs, thus suggesting NMB may modulate sensory processing, such as itch, in NHPs. Regardless of the exact neuroanatomical site releasing endogenous ligands, it is important to understand that NMB and GRP are released within the SDH, and subsequently mediate itch sensation. I.t. GRP elicited robust scratching behavior, whereas NMB caused mild scratching behavior. The expression patterns of NMB and NMBR in comparison with GRP and GRPR in NHPs are similar to a previous study performed in dogs [48]. Possibly, lower expression levels of NMBR relative to GRPR and shorter half-life of neuromedin peptides may explain these behavioral differences in scratching [49]. Higher expression level of NMB in the peripheral tissues may indicate that this peptide is involved in other peripheral functions such as muscle contraction and endocrine secretion [12,13]. Moreover, the majority of NMBR and GRPR are distributed on different subpopulations, and rarely observed in PKCγ⁺ pain-associated neurons. Therefore, NMB-NMBR and GRP-GRPR pairs in spinal cord may not significantly contribute to pain in primates.

Itch-associated functions of NMB have been determined in rodents, as the i.t. or intracerebroventricular administration of NMB increases scratching behavior. Although i.t.-administered agents could be distributed in the DRG [50], the likelihood of i.t. NMB acting on NMBR in the DRG seems low as NMBR is expressed in the DRG of NHPs at a minimal level compared to the SDH. This may indicate a species difference considering that i.t. NMB affected the NMBR-expressing DRG neurons in rodents [51]. Scratching responses by centrally administered NMB were considerably lower than those of GRP [14,16,17]. Nevertheless, another group demonstrated that NMB-induced scratching responses were significantly higher than those of GRP, based on a single dose in mice [15,19]. Different mouse strains or species may contribute to the aforementioned discrepancy. Herein, the full dose-response curve confirmed the mild scratching effects of NMB, compared with GRP in the same group of NHPs, consistent with previous rodent studies [14,16,17,52]. NMB and GRP bind to NMBR and GRPR, respectively; however, the cross-reactivity of NMB and GRP against other receptors (i.e., GRP to NMBR) constitute the cross-binding theory for itch regulation in rodents [15]. In primates, NMB-induced scratching responses were blocked by a NMBR antagonist, but not a GRPR antagonist, and vice versa, consistent with other rodent studies [16]. Therefore, each ligand (NMB and GRP) acts on its cognate receptor to elicit scratching. Unlike substance P, i.t. NMB and GRP did not affect pain sensitivity in NHPs, despite NMB-induced thermal hypersensitivity in rodents [18]. I.t. GRP did not exert anti-hypersensitive effects on carrageenan-induced thermal hyperalgesia in NHPs [6]. Furthermore, we tested if NMBR and GRPR antagonists affected thermal hypersensitivity. Normally, μ-opioid receptor agonists, such as DAMGO and morphine, induce significant antinociceptive and antiallodynic effects in rodents and primates [53]. Unlike DAMGO, neither NMBR nor GRPR antagonists attenuated capsaicin-induced allodynia. Therefore, the spinal NMB-NMBR system partially contributed to itch; however, it did not affect pain regulation in NHPs.

Behaviorally, NMB and GRP only acted on their cognate receptors for regulating itch in NHPs. Anatomically, NMB-saporin only ablated NMBR⁺ neurons, but not GRPR⁺ neurons, and vice versa in rodents [15,18]. In NHPs, the localization of NMBR and GRPR were restricted to the NeuN⁺ neurons. Moreover, the aforementioned receptors were principally expressed in VGLUT2⁺ excitatory interneurons, but not in VGAT⁺ inhibitory neurons. Therefore, both NMB-NMBR and GRP-GRPR systems directly facilitated itch transmission in the SDH. The majority of NMBR did not colocalize with GRPR, a marker of itch-responsive neurons, in the SDH of both rodents and NHPs. Thus, NMB-NMBR and GRP-GRPR systems may activate the spinal transmission of itch via independent mechanisms. Particularly, the spinal NMB-NMBR system plays a functional role in regulating mild itch.

The pharmacological effects of ligand-receptor systems vary between rodents and primates [23,24]. Particularly, opioid ligands (i.e., μ-opioid receptor and nociceptin receptor agonists) often exert diverse effects in sensory processing between rodents and primates [54,55]. μ-opioid receptor agonists, such as morphine, produces itch sensation in humans and exerts robust scratching behavior in NHPs [36,56]. However, the degree of scratching behavior caused by μ-opioid receptor agonists in rodents is considerably less than that in NHPs [16,27], thereby indicating itch sensation elicited by μ-opioid receptor agonists in humans is modeled in NHPs, but not in rodents [23]. Moreover, nociceptin receptor agonists demonstrate differential effects between rodents and NHPs. The effects of an endogenous nociceptin receptor agonist, nociceptin/orphanin FQ, on pain regulation in rodents depends on the administration route or pain modality [24,55], whereas nociceptin/orphanin FQ exerts distinct antinociceptive effects in NHPs under varied experimental conditions [25,54]. Therefore, pharmacological and behavioral studies in NHPs are pivotal for the translational relevance in humans [23]. GRP-induced scratching behavior is robust in both rodents and primates; thus, it may be reasonable to emphasize the functional and translational significance of GRP and explore the neurobiological mechanisms of itch mediated by the GRP-GRPR system in rodents. In contrast, i.t. NMB caused mild scratching behavior in NHPs; however, the potency was < 1/100, compared with that of GRP. Moreover, intradermal NMB resulted in weak scratching behavior in NHPs [51]. Taken together, NMB is highly expressed in the DRG and SDH, but the NMB-NMBR pair plays a minimal “functional” role in regulating itch than the GRP-GRPR pair in primates. Both NMBR⁺ and GRPR⁺ neurons are mainly expressed in laminae I-III of SDH, which may not explain the observed differences between GRP and NMB in NHP behavioral tests. Further studies are warranted to elucidate the other functions of NMB in the peripheral tissues and central nervous system [12].

By focusing the translational relevance of the spinal NMB-NMBR system previously evaluated only in rodents, our study revealed the neuroanatomical and functional evidence for the NMB-NMBR system in primates. Spinal NMB-NMBR and GRP-GRPR systems can independently elicit scratching; nonetheless, both systems are insignificantly involved in pain regulation. In terms of the expression patterns of NMB and NMBR in the DRG and SDH, our findings for NHPs were consistent with previous studies in rodents. Despite the high expression of NMB in DRG and SDH, low expression of NMBR in the GRPR⁺ neurons of the SDH may limit its function in regulating itch. Unlike the functional significance of spinal GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch. This warrants future neuropharmacological studies assessing translational properties from rodents to primates to validate the functional significance of these target molecules.