Fig 4 - uploaded by Loren J Martin
Content may be subject to copyright.
Altered anti-allodynic potency and efficacy of nicotine in Chrna6 mutant mice. (A to H) Dose-response relationships for the ability of systemic [intraperitoneal (i.p.); A and B], intracerebroventricular (i.c.v.; C and D), intrathecal (i.t.; E and F), and peripheral [intraplantar (i.pl.); G and H] nicotine to reverse already developed (and maximal) mechanical allodynia produced by SNI (day 7 after surgery; A, C, E, and G) and CFA (day 3 after injection; B, D, F, and H). Symbols (n = 4 to 8 mice per dose per genotype) represent mean ± SEM percentage of maximum possible anti-allodynia, based on the pre-SNI/CFA and post-SNI/CFA withdrawal thresholds of each mouse (see Materials and Methods). Statistical analyses are shown in table S3.

Altered anti-allodynic potency and efficacy of nicotine in Chrna6 mutant mice. (A to H) Dose-response relationships for the ability of systemic [intraperitoneal (i.p.); A and B], intracerebroventricular (i.c.v.; C and D), intrathecal (i.t.; E and F), and peripheral [intraplantar (i.pl.); G and H] nicotine to reverse already developed (and maximal) mechanical allodynia produced by SNI (day 7 after surgery; A, C, E, and G) and CFA (day 3 after injection; B, D, F, and H). Symbols (n = 4 to 8 mice per dose per genotype) represent mean ± SEM percentage of maximum possible anti-allodynia, based on the pre-SNI/CFA and post-SNI/CFA withdrawal thresholds of each mouse (see Materials and Methods). Statistical analyses are shown in table S3.

Source publication
Article
Full-text available
Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subuni...

Contexts in source publication

Context 1
... = 0.01 and t 16 = 2.2, P < 0.05, respectively). There were no significant genotype × sex interactions ob- served in any data set. In an experiment performed independently, in a different laboratory, using Chrna6 KO mice and another neuropathic assay [chronic constriction injury (CCI)], the increased mechanical allodynia of KO mice was confirmed ( fig. S4A). A separate head-to-head exper- iment using CCI and CFA in Chrna6 and Chrna4 KO mice confirmed the significantly increased allodynia in Chrna6 KOs, but revealed no differences between Chrna4 KOs and their WT controls ( fig. S4, B and C). The a6 subunit appears to play a highly specific role in the modulation of mechanical allodynia ...
Context 2
... neuropathic assay [chronic constriction injury (CCI)], the increased mechanical allodynia of KO mice was confirmed ( fig. S4A). A separate head-to-head exper- iment using CCI and CFA in Chrna6 and Chrna4 KO mice confirmed the significantly increased allodynia in Chrna6 KOs, but revealed no differences between Chrna4 KOs and their WT controls ( fig. S4, B and C). The a6 subunit appears to play a highly specific role in the modulation of mechanical allodynia because Chrna6 KO mice showed statistically equivalent responses to WT mice on a battery of acute and tonic nociceptive assays ( fig. ...
Context 3
... intrathecal, and peripheral (intraplantar) (−)-nico- tine to reverse mechanical allodynia produced by either SNI or CFA in WT, KO, and L9′S mice. Although potency and efficacy varied by route of administration, nicotine was significantly and dose-dependently ef- fective against both types of allodynia in WT mice by all injection routes ( Fig. 4 and table S3). Gain-of-function L9′S mutants showed similar or significantly increased efficacy, but Chrna6 KO mice dis- played no significant nicotine-induced anti-allodynia in either assay by any route. We then performed a head-to-head comparison of supra- spinal, spinal, and peripheral nicotine-induced anti-allodynia (25 mg, ...
Context 4
... (n = 5 to 12 mice per genotype) represent mean ± SEM paw withdrawal threshold (g) on each testing day; bars represent mean ± SEM percentage of maximum possible allodynia (see Materials and Methods). *P < 0.05, **P < 0.01, ***P < 0.001 compared to other genotype. A replication of the KO data, using a different neuropathic assay, can be found in fig. S4. at any concentration. We found as well that P2X2, P2X3, or P2X2/3 currents were not affected by ACh (100 mM). In four of the six cases where we could study dose-response relations, we found only minor (less than two-fold) changes in the EC 50 (median effective concentra- tion) values, and insignificant changes in the Hill coefficient, ...
Context 5
... to von Frey fiber stimulation measured before (−14, −7 days) and after (1,4,7,14,21, and 28 days) SNI surgery. Fig. S2. Sex differences in Chrna6 DRG mRNA expression in SM mice and their correlation with sex differences in mechanical allodynia. Fig. S3. Down-regulation of Chrna6 by nerve injury and correlation with mechanical allodynia. Fig. S4. Increased ipsilateral mechanical allodynia in Chrna6 (a6 KO) but not Chrna4 null mu- tants (a4 KO). Fig. S5. No altered sensitivity of Chrna6 KO mice in a battery of acute and tonic nociceptive assays. Fig. S6. Physical contacts between P2X3 and a6b4* nAChRs revealed by FLIM. Table S1. Affymetrix gene expression data of all Chrn* ...

Similar publications

Article
Full-text available
Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect caused by chemotherapy drugs, and its existence seriously affects the quality of life of patients. We first established an oxaliplatin-induced peripheral neuropathy (OIPN) model and then measured and evaluated mechanical hyperalgesia, thermal nociception, cold allodyni...
Article
Full-text available
Introduction After excision of a primary malignant melanoma (MM), treatment of stage IB or higher MM consists of sentinel lymph node biopsy (SLNB). If malignant cells are identified, a complete lymph node dissection (CLND) can be performed. Objective To determine the natural history of pain and sensory changes after MM surgery. Methods We prospec...
Article
Full-text available
Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α1...
Article
Full-text available
Background Alternative medicine is noted for its clinical effect and minimal invasiveness in the treatment of neuropathic pain. Go-sha-jinki-Gan, a traditional Japanese herbal medicine, has been used for meralgia and numbness in elderly patients. However, the exact mechanism of GJG is unclear. This study aimed to investigate the molecular mechanism...
Article
Full-text available
The management of persistent postsurgical pain and neuropathic pain remains a challenge in the clinic. Local anesthetics have been widely used as simple and effective treatment for these two disorders, but the duration of their analgesic effect is short. We here reported a new poly lactic-co-glycolic acid (PLGA)-coated ropivacaine that was continuo...

Citations

... Previous research has reported this receptor regulating exocytosis in human adrenal chromaffin cells [11,12]. Besides, α6β4* nAChRs are highly expressed in dorsal root ganglia neurons, which are involved in pain [13][14][15]. Therefore, α6β4* nAChR has the potential to be developed as an attractive non-opioid therapeutic target for pain. ...
... α3β2 nAChRs were found in the dorsal root ganglia, which is associated with regulating pain awareness [24]. α6β4* is another type of nAChR also highly expressed by the dorsal root ganglia, which is an attractive target for treating neuropathic pain [13,15,25]. Furthermore, α6β4* nAChR is also found in the hippocampus regulating the secretion of norepinephrine, which is closely related to learning and memory [15,26]. ...
Article
Full-text available
α6β4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6β4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3β2 and α6/α3β4 nAChR subtypes with significantly high affinity (IC50 was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 μM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3β4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6β2β3 and/or α3β4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6β4 nAChR.
... We found that three genes coding subunits for nicotinic acetylcholine receptors, chrna1, chrna2b and chrna6, are downregulated in enteric neurons in absence of chd8. Mutations in CHRNA1, and CHRNA6 have been implicated in fast-channel congenital myasthenic syndrome (MIM#608930) characterized by early-onset progressive muscle weakness, and chronic pain 53,54 . Of . ...
Preprint
Full-text available
Gastrointestinal complaints in autism are common and impact the quality of life of affected individuals, yet the underlying mechanisms are understudied. We have found that individuals with mutations in CHD8 present with gastrointestinal disturbances. We have shown that loss of chd8, the sole ortholog of CHD8 in zebrafish, leads to reduced number of enteric neurons and decreased intestinal mobility. However, it remains unclear how chd8 acts during the development of the enteric nervous system and whether CHD8-associated gastrointestinal complaints are solely due to impaired neuronal function in the intestine. Here, utilizing a stable chd8 mutant zebrafish model, we found that the loss of chd8 leads to reduced number of vagal neural crest cells (NCCs), enteric neural progenitors, emigrating from the neural tube and their early migration capability was altered. At later stages, although the intestinal colonization by the NCCs was complete, we found decreased numbers of both NCC-derived serotonergic neurons and serotonin-producing enterochromaffin cells, suggesting an intestinal hyposerotonemia in absence of chd8. Moreover, transcriptomic analyses revealed altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. Next, tissue examination of chd8 mutants revealed thinner intestinal epithelium accompanied by accumulation of neutrophils and decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole mid- and posterior intestines showed a global disruption of the immune balance with perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, serotonergic pathway, intestinal homeostasis, and autism-associated gastrointestinal complaints.
... Another pharmacologically important a6containing receptor is a diheteromer containing the b4 subunit that occurs in the sensory neurons of dorsal root ganglia (58). BARP and IRE1a are needed to reconstitute a6b4 receptors, which are curiously not affected by NACHO (59). ...
... Unfortunately, ABT-594 had unacceptable adverse effects (88), and its therapeutic nAChR target was unknown. Subsequently, a genomics screen of dorsal root ganglion tissue from outbred mouse strains identified that a6 nAChR subunit mRNA levels inversely correlate with pain responses in a spared nerve injury model (58). Accordingly, analgesic effects of nicotine after both inflammatory and neuropathic injuries are absent in a6 KO mice. ...
... Accordingly, analgesic effects of nicotine after both inflammatory and neuropathic injuries are absent in a6 KO mice. Furthermore, human postoperative pain and temporomandibular disorder are affected by a polymorphism in the CHRNA6 (a6) promoter (58). ...
Article
The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR–specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.
... On the other side, the reduced expression of β2 could contribute to the allodoynic phenotype of cuff mice, as we have previously shown that β2-knock out mice have a decreased mechanical threshold (Yalcin et al., 2011b). This is reminiscent of the negative correlation observed between the mRNA level of the α6 nAChR subunit (variable across inbred mice strains) and the amount of allodynia the mice develop after spare-nerve injury (Wieskopf et al., 2015). In contrast with our results, a study in rats using a peripheral axotomy model found an increase in the mRNA levels for α5 and β2 in the axotomy dorsal horn vs. control (Yang et al., 2004). ...
Article
Full-text available
Endogenous acetylcholine (ACh) is an important modulator of nociceptive sensory processing in the spinal cord. An increased level of spinal ACh induces analgesia both in humans and rodents while interfering with cholinergic signaling is allodynic, demonstrating that a basal tone of spinal ACh modulates nociceptive responses in naïve animals. The plasticity undergone by this cholinergic system in chronic pain situation is unknown, and the mere presence of this tone in neuropathic animals is controversial. We have addressed these issues in mice through behavioral experiments, histology, electrophysiology and molecular biology, in the cuff model of peripheral neuropathy. Our behavior experiments demonstrate the persistence, and even increased impact of the analgesic cholinergic tone acting through nicotinic receptors in cuff animals. The neuropathy does not affect the number or membrane properties of dorsal horn cholinergic neurons, nor specifically the frequency of their synaptic inputs. The alterations thus appear to be in the neurons receiving the cholinergic signaling, which is confirmed by the fact that subthreshold doses of acetylcholinesterase (AChE) inhibitors in sham animals become anti-allodynic in cuff mice and by the altered expression of the β2 nicotinic receptor subunit. Our results demonstrate that endogenous cholinergic signaling can be manipulated to relieve mechanical allodynia in animal models of peripheral neuropathy. Until now, AChE inhibitors have mainly been used in the clinics in situations of acute pain (parturition, post-operative). The fact that lower doses (thus with fewer side effects) could be efficient in chronic pain conditions opens new avenues for the treatment of neuropathic pain. Significance statement Chronic pain continues to be the most common cause of disability that impairs the quality of life, accruing enormous and escalating socio-economic costs. A better understanding of the plasticity of spinal neuronal networks, crucially involved in nociceptive processing, could help designing new therapeutic avenues. We here demonstrate that chronic pain modifies the spinal nociceptive network in such a way that it becomes more sensitive to cholinergic modulations. The spinal cholinergic system is responsible for an analgesic tone that can be exacerbated by acetylcholinesterase inhibitors, a property used in the clinic to relief acute pain (child birth, post-op). Our results suggest that lower doses of acetylcholinesterases, with even fewer side effects, could be efficient to relieve chronic pain.
... It will be interesting to see, in future work, how Prdm12 and Ehmt2 potentially synergistically affect transcriptional changes after injury. Second, overexpression of Chrna6, which encodes the a6 subunit of the nicotinic acetylcholine receptor (nAChR), is protective against tactile allodynia in both neuropathic and inflammatory injury models (Wieskopf et al., 2015). Chrna6 was also the only gene found to be upregulated in Prdm12 AvilERT2CKO mice, about 2-fold over control levels. ...
Article
Full-text available
Prdm12 is a key transcription factor in nociceptor neurogenesis. Mutations of Prdm12 cause congenital insensitivity to pain (CIP) from failure of nociceptor development. However, precisely how deletion of Prdm12 during development or adulthood affects nociception is unknown. Here, we employ tissue- and temporal-specific knockout mouse models to test the function of Prdm12 during development and in adulthood. We find that constitutive loss of Prdm12 causes deficiencies in proliferation during sensory neurogenesis. We also demonstrate that conditional knockout from dorsal root ganglia (DRGs) during embryogenesis causes defects in nociception. In contrast, we find that, in adult DRGs, Prdm12 is dispensable for most pain-sensation and injury-induced hypersensitivity. Using transcriptomic analysis, we find mostly unique changes in adult Prdm12 knockout DRGs compared with embryonic knockout and that PRDM12 is likely a transcriptional activator in the adult. Overall, we find that the function of PRDM12 changes over developmental time.
... Innervation of mouse gut by DRG neurons is provided by ganglia located at levels T8-L1 and L6-S1 [29] and have been shown to express α4β2*, α7, and α3β4* nAChRs based on receptor sensitivities to subtype-selective antagonists [30,31]. The functional role of nAChRs in DRG neurons is poorly understood, but α3β4*, α6β4*, and α7 nAChRs have been reported to be expressed by putative nociceptors and may therefore be involved in nociception [28,32,33]. Additionally, α7 nAChRs located on DRG neuron terminals in the dorsal horn of the spinal cord modulate the release of glutamate and have been proposed to be involved in nicotine mediated analgesia [34]. ...
Article
Full-text available
The gut-brain axis describes a complex interplay between the central nervous system and organs of the gastrointestinal tract. Sensory neurons of dorsal root and nodose ganglia, neurons of the autonomic nervous system, and immune cells collect and relay information about the status of the gut to the brain. A critical component in this bi-directional communication system is the vagus nerve which is essential for coordinating the immune system’s response to the activities of commensal bacteria in the gut and to pathogenic strains and their toxins. Local control of gut function is provided by networks of neurons in the enteric nervous system also called the ‘gut-brain’. One element common to all of these gut-brain systems is the expression of nicotinic acetylcholine receptors. These ligand-gated ion channels serve myriad roles in the gut-brain axis including mediating fast synaptic transmission between autonomic pre- and postganglionic neurons, modulation of neurotransmitter release from peripheral sensory and enteric neurons, and modulation of cytokine release from immune cells. Here we review the role of nicotinic receptors in the gut-brain axis with a focus on the interplay of these receptors with the gut microbiome and their involvement in dysregulation of gut function and inflammatory bowel diseases.
... Recent studies are now starting to delineate roles for other nAChRs subtypes in a number of diseases. For example, despite the limited neuronal distribution of α6β2 * subtypes, expression of the α6 subunit in nociceptors suggests it could contribute to sensory processing and pain (Hone and McIntosh, 2018), with an inverse correlation between CHRNA6 expression and neuropathic pain found in mice and humans (Wieskopf et al., 2015). More recently, the α9 * has also been implicated in modulating the pathophysiology of neuropathic pain (Hone and McIntosh, 2018;Hone et al., 2018a). ...
Article
Full-text available
Neuronal nicotinic acetylcholine receptors (nAChRs) are prototypical cation-selective, ligand-gated ion channels that mediate fast neurotransmission in the central and peripheral nervous systems. nAChRs are involved in a range of physiological and pathological functions and hence are important therapeutic targets. Their subunit homology and diverse pentameric assembly contribute to their challenging pharmacology and limit their drug development potential. Toxins produced by an extensive range of algae, plants and animals target nAChRs, with many proving pivotal in elucidating receptor pharmacology and biochemistry, as well as providing templates for structure-based drug design. The crystal structures of these toxins with diverse chemical profiles in complex with acetylcholine binding protein (AChBP), a soluble homolog of the extracellular ligand-binding domain of the nAChRs and more recently the extracellular domain of human α9 nAChRs, have been reported. These studies have shed light on the diverse molecular mechanisms of ligand-binding at neuronal nAChR subtypes and uncovered critical insights useful for rational drug design. This review provides a comprehensive overview and perspectives obtained from structure and function studies of diverse plant and animal toxins and their associated inhibitory mechanisms at neuronal nAChRs.
... The purpose of these trainings are to reassure the patients, explain parafunctional nature and its possible causes. Early disease detection and ultimately reducing repetitive muscle tensions such as teeth grinding, encourage and strengthen the vulnerable individuals, teach them relaxation and control related muscle activities [23]. Various causes and factors that may have been involved in the development of Brux-ism have been discussed, however the genetic factor contribution is also emphasized and confirmed, and as a result some researchers and even psychologists characterize several genes for the expression of a unique or specific existence with bruxism in anxious individuals and individuals with neurological disorders; indicating association with specific polymorphism rs6313 of the 5HT2A gene [24,25]. ...
Article
Background: Bruxism is a parafunctional disorder. The prevalence of this rhythmic activity of rodent muscles is reported to be about %8. This disease can compromise the life quality of a person’s general performance. The aim of this study is to gather information upon genetic factors, which contribute to the pathogenesis of the disease. Materials and Methods: All related articles published in 1966 onward from google scholar such as ISI, PubMed, Scopus and Ovid within the databases were searched using English keywords ‘Bruxism and Genetics’. 300 articles were found. 252 articles were removed due to content duplication and irrelevance. Results: The review of selected articles finally showed that in addition to other factors such as psychological factors, local factors, systemic factors, etc., the genetic factors also play a significant role in pathogenesis of bruxism. Among the influential genes are rs6313 polymorphism from the 5HT2A gene and rs6313 polymorphism from the HTR2A gene. Conclusion: Evidence suggests that genetic factors play an important role in the pathology and development of bruxism, however the main causing mechanism still largely remains unknown. Keywords: Genetic factors; Bruxism; Article review; Polymorphism.
... Based on our previous work, we conclude alpha6/alpha3 nicotinic receptors in these neuronal groups are down-regulated after nerve injury. Loss of alpha6 nicotinic receptors have been shown to increase pain responses (18) and, thus, the loss of nicotinic receptors in these two groups of nonpeptidergic neurons can be relevant to the establishment of chronic pain states. The bradykinin responsive cell groups were analyzed for these stains. ...
Article
Full-text available
Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.
... While α6β4 in DRG neurons plays a key role in nicotine-induced antiallodynia, dopaminergic neurons of the brain express an especially varied collection of other nAChR subtypes. The Janssen Pharmaceutical group recently identified accessory proteins that increase the protein levels of resembles that of α4β2 nAChRs, it has also been suspected that α6-containing nAChRs are also targets for nAChR agonists' analgesic properties (13). The Gordian knot has been achieving functional expression of α6-containing nAChRs. ...
Article
Useful animal models of disease in neuroscience can make accurate predictions about a therapeutic outcome, a feature known as predictive validity. In this issue of the JCI, Knowland et al. provide an improved model to assess nicotinic acetylcholine receptor (nAChR) ligands for treating chronic pain. The authors identify two proteins, the voltage-dependent calcium channel auxiliary subunit BARP and the unfolded protein response sensor IRE1α, that are required for robust heterologous expression of α6β4, an nAChR subtype in dorsal root ganglia (DRG). This nAChR is a candidate for the analgesic effects of nicotine as well as the frog toxin epibatidine. Now researchers can efficiently screen for α6β4 nAChR–selective agonists using heterologous expression systems. Candidates that emerge will enable researchers to test the predictive validity of mouse models for chronic pain in the nAChR context. If all these steps work, one can envision a class of non-opioid nAChR-targeted analgesics for chronic pain.