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![The relationship of thyrotropin-releasing hormone (TRH)-expressing dorsal horn neurons to cholecystokinin (CCK), protein kinase Cγ (PKCγ), and neurotensin. (a-c) A scan through laminae II and III from a section reacted with probes against TRH, CCK, and PKCγ mRNAs, and counterstained with NucBlue. Three TRH+ cells are marked with arrows, and all of these are positive for both CCK and PKCγ mRNAs. Asterisks indicate two PKCγ cells that lack message for TRH and CCK, while the arrowheads show two CCK cells that are negative for TRH and PKCγ. (d-f) A scan through the equivalent region from a section reacted with probes against TRH, neurotensin, and PKCγ mRNAs, and counterstained with NucBlue. Two TRH+ cells that contain mRNA for PKCγ, but not for neurotensin, are indicated with arrows. The arrowheads point to two cells that are positive for neurotensin and PKCγ mRNA, but negative for TRH mRNA. All images were obtained from single optical sections. Scale bar = 20 μm [Color figure can be viewed at wileyonlinelibrary.com]](publication/330966873/figure/fig6/AS:735326641336323@1552326941500/The-relationship-of-thyrotropin-releasing-hormone-TRH-expressing-dorsal-horn-neurons-to.jpg)
The relationship of thyrotropin-releasing hormone (TRH)-expressing dorsal horn neurons to cholecystokinin (CCK), protein kinase Cγ (PKCγ), and neurotensin. (a-c) A scan through laminae II and III from a section reacted with probes against TRH, CCK, and PKCγ mRNAs, and counterstained with NucBlue. Three TRH+ cells are marked with arrows, and all of these are positive for both CCK and PKCγ mRNAs. Asterisks indicate two PKCγ cells that lack message for TRH and CCK, while the arrowheads show two CCK cells that are negative for TRH and PKCγ. (d-f) A scan through the equivalent region from a section reacted with probes against TRH, neurotensin, and PKCγ mRNAs, and counterstained with NucBlue. Two TRH+ cells that contain mRNA for PKCγ, but not for neurotensin, are indicated with arrows. The arrowheads point to two cells that are positive for neurotensin and PKCγ mRNA, but negative for TRH mRNA. All images were obtained from single optical sections. Scale bar = 20 μm [Color figure can be viewed at wileyonlinelibrary.com]
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Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non‐overlapping populations in laminae I‐III, based on expression of substance P, gastrin‐releasing peptide, neurokinin B and neurotensin. Cholecystokinin (CCK) is expressed by many dor...
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The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin,...
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... Cholecystokinin B receptor (CCKBR) and cholecystokinin (CCK) in sensory ganglia, spinal cord, and supraspinal neurons mediate nociception, morphine insensitivity, and anxiety in numerous rodent pain models [40][41][42][43][44][45]. CCK was elevated in DRG two weeks post-sciatic nerve transection and was correlated with morphine insensitivity [41]. ...
The information provided from the papers reviewed here about the role of epigenetics in chronic craniofacial neuropathic pain is critically important because epigenetic dysregulation during the development and maintenance of chronic neuropathic pain is not yet well characterized, particularly for craniofacial pain. We have noted that gene expression changes reported vary depending on the nerve injury model and the reported sample collection time point. At a truly chronic timepoint of 10 weeks in our model of chronic neuropathic pain, functional groupings of genes examined include those potentially contributing to anti-inflammation, nerve repair/regeneration, and nociception. Genes altered after treatment with the epigenetic modulator LMK235 are discussed. All of these differentials are key in working toward the development of diagnosis-targeted therapeutics and likely for the timing of when the treatment is provided. The emphasis on the relevance of time post-injury is reiterated here.
... We also observed co-expression of certain neuropeptide genes such as NMU and TAC3 (Tac2 in mice) in lamina II, which has also been reported for mice. One exception we observed was a higher percentage of CCK expressing cells in the superficial laminae of humans compared to what we observed in macaques and what has been published for mice [33][34][35] . ...
Key mechanisms underlying chronic pain occur within the neural circuitry of the dorsal horn. Recent genome-wide association studies (GWAS) have identified genetic variants associated with the predisposition to chronic pain. However, most of these variants lie in regulatory non-coding regions that have so far not been linked to spinal cord function. Here, we take a multi-species approach to determine whether chronic pain variants impact regulatory elements of dorsal horn neurons. We first built a more comprehensive single cell atlas; filling gaps by generating a high-quality Rhesus macaque atlas and integrating it with human and mouse. With cellular-resolution spatial transcriptomics, we mapped the laminar distributions of the resulting species-conserved neuron subtypes, uncovering an unexpected organization. Lastly, we generated a mouse single-nucleus open chromatin atlas to partition the heritability of chronic pain traits. From this, we identified strong, selective associations between specific, conserved neuron subtypes and major forms of chronic pain.
... CCK-BR is involved in several different aspects of the human pain experience that are particularly prominent in females [9]. CCKBR and its neuropeptide ligand, CCK, are widely expressed in the sensory ganglia, spinal cord, and brain pain circuitry [10,11]. Understanding how an antibody interacts with its targets is critical for the development of that antibody as a therapeutic drug. ...
... CCK-BR is involved in several different aspects of the human pain experience that are particularly prominent in females [9]. CCKBR and its neuropeptide ligand, CCK, are widely expressed in the sensory ganglia, spinal cord, and brain pain circuitry [10,11]. Axotomy results in CCK upregulation in sensory neurons (30%) after 14 days [10]. ...
... CCK-BR is involved in several different aspects of the human pain experience that are particularly prominent in females [9]. CCKBR and its neuropeptide ligand, CCK, are widely expressed in sensory ganglia, spinal cord, and the brain pain circuitry [10,11]. Axotomy results in CCK upregulation in sensory neurons (30%) after 14 days [10]. ...
A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10–8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.
... For example, Grudt and Perl 9 combined whole-cell patch-clamp recording with morphological reconstruction and described 3 classes in lamina II (vertical, radial and transient central cells) that were subsequently shown to be excitatory 12,13 . We have recently identified 7 largely non-overlapping neurochemical populations among SDH excitatory interneurons [13][14][15][16][17][18] . Five of these are defined by expression of a neuropeptide (neurotensin, neurokinin B, cholecystokinin, substance P or neuropeptide FF), and one by the presence of gastrin-releasing peptide receptor mRNA (for convenience we refer to these as GRPR cells). ...
Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75–80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.
... Five of these are defined by expression of cholecystokinin (CCK), neurotensin, neurokinin B (NKB), substance P (SP), and neuropeptide FF (NPFF), and the sixth by the presence of green fluorescent protein (GFP) in a GRP::GFP transgenic mouse line. 7,23,25,26 These classes account for ;75% of excitatory interneurons in laminae I-II and show good correspondence with transcriptomic populations. 27 We reported that the SP and GRP-GFP populations include radial and transient central cells, respectively, whereas the CCK, neurotensin, and NKB populations overlap extensively with PKCg cells in the inner part of lamina II and lamina III. ...
... 27 We reported that the SP and GRP-GFP populations include radial and transient central cells, respectively, whereas the CCK, neurotensin, and NKB populations overlap extensively with PKCg cells in the inner part of lamina II and lamina III. 16,25 Another population of excitatory interneurons that has attracted considerable attention consists of cells expressing the gastrin-releasing peptide receptor (GRPR). These are located in lamina I and outer lamina II, and there is strong evidence implicating them in itch. ...
... Positive and negative control probes were also tested on other sections, as described previously. 25 Sections were mounted with ProLong Glass antifade medium with NucBlue (Hoechst 33342; Thermo Fisher Scientific, Paisley, United Kingdom). The full thickness of the section was scanned with a Zeiss LSM 710 confocal microscope equipped with Argon multiline, 405-nm diode, 561-nm solid state, and 633-nm HeNe lasers, through a 403 oil immersion lens (numerical aperture, NA, 1.3), and tile scanning was used to cover the whole of laminae I-II. ...
Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are thought to transmit nociceptive information. In this study, we have used a GRPRCreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the great majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain- and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.
... Thus, peripheral CCK2R is otherwise known as the "gastrin receptor". In the nervous system CCKRs are distributed in a variety of areas including the cortex, amygdala, anterior cingulate cortex (ACC), substantia nigra, ventral tegmental area (VTA), periaqueductal grey (PAG), rostral ventromedial medulla (RVM), and the spinal cord (Hill et al., 1987;Mercer and Beart, 2004;Gutierrez-Mecinas, 2019;Sjöstedt, 2020). Within the spinal cord, CCK + neurons are known to play a role in mechanical allodynia in both inflammatory and neuropathic models (Peirs et al., 2021). ...
The cholecystokinin receptor system, specifically cholecystokinin 2 receptor (CCK 2R) is a historic target for pain management that has shown limited success. However, new approaches to target CCK2R have incited fresh enthusiasm for this target. In this mini-review, we discuss what is known about CCK2R in peripheral and central circuits under naïve physiological conditions and under conditions of chronic pain, the interactions of CCK2Rs with opioids and briefly, recent efforts to develop new treatments targeting CCK2R for chronic pain.
... The excitatory and inhibitory neurons in the spinal dorsal horn have been morphologically and electrophysiologically analyzed, and their subtypes have also been identified by molecular and gene expression profiles [6][7][8][9][10][11]. These findings promote the understanding of neuroplasticity occurring in interneurons of the spinal dorsal horn circuit, which is associated with allodynia after PNS injury. ...
Neuropathic pain is often chronic and can persist after overt tissue damage heals, suggesting that its underlying mechanism involves the alteration of neuronal function. Such an alteration can be a direct consequence of nerve damage or a result of neuroplasticity secondary to the damage to tissues or to neurons. Recent studies have shown that neuroplasticity is linked to causing neuropathic pain in response to nerve damage, which may occur adjacent to or remotely from the site of injury. Furthermore, studies have revealed that neuroplasticity relevant to chronic pain is modulated by microglia, resident immune cells of the central nervous system (CNS). Microglia may directly contribute to synaptic remodeling and altering pain circuits, or indirectly contribute to neuroplasticity through property changes, including the secretion of growth factors. We herein highlight the mechanisms underlying neuroplasticity that occur in the somatosensory circuit of the spinal dorsal horn, thalamus, and cortex associated with chronic pain following injury to the peripheral nervous system (PNS) or CNS. We also discuss the dynamic functions of microglia in shaping neuroplasticity related to chronic pain. We suggest further understanding of post-injury ectopic plasticity in the somatosensory circuits may shed light on the differential mechanisms underlying nociceptive, neuropathic, and nociplastic-type pain. While one of the prominent roles played by microglia appears to be the modulation of post-injury neuroplasticity. Therefore, future molecular- or genetics-based studies that address microglia-mediated post-injury neuroplasticity may contribute to the development of novel therapies for chronic pain.
... Blockade of CCK 2 receptors reduces anxiety-induced hyperalgesia in humans (Benedetti et al., 2006). It has been demonstrated that CCK expressing cells in the dorsal horn of the spinal cord have important roles in mechanical allodynia in neuropathic pain states (Gutierrez-Mecinas et al., 2019). However, whether spinal CCK contributes to the development of the comorbidity of TMD and FMS remains unclear. ...
In some chronic primary pain conditions such as temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS), mild or chronic stress enhances pain. TMD and FMS often occur together, but the underlying mechanisms are unclear. The purpose of this study was to investigate the role of cholecystokinin (CCK) in the spinal cord in somatic hyperalgesia induced by orofacial inflammation combined with stress. Somatic hyperalgesia was detected by the thermal withdrawal latency and mechanical withdrawal threshold. The expression of CCK1 receptors, CCK2 receptors, ERK1/2 and p-ERK1/2 in the spinal cord was examined by Western blot. After the stimulation of orofacial inflammation combined with 3 day forced swim, the expression of CCK2 receptors and p-ERK1/2 protein in the L4-L5 spinal dorsal horn increased significantly, while the expression of CCK1 receptors and ERK1/2 protein remained unchanged. Intrathecal injection of the CCK2 receptor antagonist YM-022 or mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked somatic hyperalgesia induced by orofacial inflammation combined with stress. Intrathecal administration of the MEK inhibitor blocked somatic sensitization caused by the CCK receptor agonist CCK8. The CCK2 receptor antagonist YM-022 significantly reduced the expression of p-ERK1/2. These data indicate that upregulation of CCK2 receptors through the MAPK pathway contributes to somatic hyperalgesia in this comorbid pain model. Thus, CCK2 receptors and MAPK pathway may be potential targets for the treatment of TMD comorbid with FMS.
... Three wild-type C57BL/6 mice (both sexes, 18-20 g) were used for in situ hybridization experiments, as described previously. 36 Animals were decapitated under deep isoflurane anaesthesia before their spinal cords were removed and then rapidly frozen on dry ice. Fresh frozen lumbar spinal cord segments were embedded in OCT mounting medium and then cut into 12-mmthick transverse sections on a cryostat (Leica CM1860; Leica, Milton Keynes, United Kingdom) and mounted on Superfrost Plus slides (48311-703; VWR, Lutterworth, United Kingdom). ...
... Semiautomated analysis of transcript numbers per nucleus was conducted using the cell detection and subcellular object features on QuPath software. 36 Cell analysis was conducted only in laminae IIi and III, where a band of dense PV transcripts was present. Recognition and segmentation of individual nuclei was performed based on NucBlue staining. ...
... 57 Given most ePVINs also express CCK, peptide co-transmission could similarly enhance postsynaptic responses to ePVIN inputs. Furthermore, CCK release has been implicated in tactile allodynia, 42 CCK receptors are expressed in several dorsal horn populations, 37 and CCK is widely distributed in mouse dorsal horn cells 36 including CCK-expressing cells lacking PKCg that are implicated in mechanical allodynia. 61 Finally, the general distribution of these CCK-expressing cells overlaps with the ePVIN population, and CCK and PV are both restricted to the same transcriptomic groupings. ...
Parvalbumin-expressing interneurons (PVINs) in the spinal dorsal horn are found primarily in laminae II inner and III. Inhibitory PVINs (iPVINs) play an important role in segregating innocuous tactile input from pain-processing circuits through presynaptic inhibition of myelinated low-threshold mechanoreceptors and postsynaptic inhibition of distinct spinal circuits. By comparison, relatively little is known of the role of excitatory PVINs (ePVINs) in sensory processing. Here we use neuroanatomical and optogenetic approaches to show that ePVINs comprise a larger proportion of the PVIN population than previously reported, and that both ePVIN and iPVIN populations form synaptic connections amongst (and between) themselves. We find that these cells contribute to neuronal networks that influence activity within several functionally distinct circuits, and that aberrant activity of ePVINs under pathological conditions is well placed to contribute to the development of mechanical hypersensitivity.
... Axotomy results in CCK upregulation in sensory neurons (30%) after 14 days (Wiesenfeld-Hallin et al., 1997;Bras et al., 1999;Xu et al., 1993). In fact, CCK-B receptor expression changes over time are contributory to chronic pain in a variety of animal models (Gutierrez-Mecinas et al., 2019;Bangash et al., 2018;Korczeniewska et al., 2018). ...
The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2-6 hours, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. A effective non-opiate, non-addictive therapy with potential to significantly reduce chronic neuropathic pain long term is greatly needed.
