Mouse Acetylcholine Receptor Research Articles

Neurobehavioral Changes in Mice Exposed to Fast Neutrons in utero

Epidemiological studies have revealed that radiation causes brain development abnormalities in atomic bomb survivors exposed in utero. Rat and mouse studies have also shown that prenatal exposure to low-linear energy transfer radiation induces developmental brain anomalies. Because the effects of prenatal irradiation on adult behavior patterns remain largely unknown, the present study investigated the effects of neutron exposure in utero on postnatal behavior patterns in mice. [C57BL/6J × C3H/He] hybrid (B6C3F1) mice were exposed to cyclotron-derived fast neutrons with peak energy of 10 MeV (0.02-0.2 Gy) or Cs-137 gamma-rays (0.2-1.5 Gy) on embryonic day 13.5. At 5.5-8 months of age, the neurobehavior of male offspring was examined by Rota-rod treadmill and locomotor activity. The accumulation of radio-labeled drug at muscarinic acetylcholine and serotonin receptors in mice from control and neutron-irradiated groups was determined by the tracer method. Locomotor activity during the dark period increased in the 0.02 Gy neutron-irradiated group. Furthermore, at 5.5 months of age, tracer binding in vivo to the muscarinic acetylcholine increased and to the serotonin receptors decreased in the 0.02 Gy neutron-irradiated group. In conclusion, the present study reveals that a certain "low-dose window" may exist for radiation-induced changes in neurobehavior and binding to neurotransmitter receptors, because there was correlation in neurobehavior and binding to neurotransmitter receptors in the 0.02 Gy neutron-irradiated group though there was not correlation in the neutron-irradiated groups more than 0.05 Gy.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Long-term effects of methamphetamine exposure on cognitive function and muscarinic acetylcholine receptor levels in mice.

Exposure to methamphetamine during brain development impairs cognition in humans and rodents. In mice, these impairments are more severe in females than males. Genetic factors, such as apolipoprotein E genotype, may modulate the cognitive effects of methamphetamine. Methamphetamine-induced alterations in the brain acetylcholine system may contribute to the cognitive effects of methamphetamine and may also be modulated by apolipoprotein E isoform. We assessed the long-term effects of methamphetamine exposure during brain development on cognitive function and muscarinic acetylcholine receptors in mice, and whether apolipoprotein E isoform modulates these effects. Mice expressing human apolipoprotein E3 or E4 were exposed to methamphetamine (5 mg/kg) or saline once a day from postnatal days 11-20 and behaviorally tested in adulthood. Muscarinic acetylcholine receptor binding was measured in the hippocampus and cortex. Methamphetamine exposure impaired novel location recognition in female, but not male, mice. Methamphetamine-exposed male and female mice showed impaired novel object recognition and increased number of muscarinic acetylcholine receptors in the hippocampus. The cognitive and cholinergic effects of methamphetamine were similar in apolipoprotein E3 and E4 mice. Thus, the cholinergic system, but not apolipoprotein E isoform, might play an important role in the long-term methamphetamine-induced cognitive deficits in adulthood.

Beneficial Metabolic Effects Caused by Persistent Activation of β-Cell M3 Muscarinic Acetylcholine Receptors in Transgenic Mice

Previous studies have shown that β-cell M(3) muscarinic acetylcholine receptors (M3Rs) play a key role in maintaining blood glucose homeostasis by enhancing glucose-dependent insulin release. In this study, we tested the hypothesis that long-term, persistent activation of β-cell M3Rs can improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet. To achieve the selective and persistent activation of β-cell M3Rs in vivo, we generated transgenic mice that expressed the Q490L mutant M3R in their pancreatic β-cells (β-M3-Q490L Tg mice). The Q490L point mutation is known to render the M3R constitutively active. The metabolic phenotypes of the transgenic mice were examined in several in vitro and in vivo metabolic tests. In the presence of 15 mm glucose and the absence of M3R ligands, isolated perifused islets prepared from β-M3-Q490L Tg mice released considerably more insulin than wild-type control islets. This effect could be completely blocked by incubation of the transgenic islets with atropine (10 μm), an inverse muscarinic agonist, indicating that the Q490L mutant M3R exhibited ligand-independent signaling (constitutive activity) in mouse β-cells. In vivo studies showed that β-M3-Q490L Tg mice displayed greatly improved glucose tolerance and increased serum insulin levels as well as resistance to diet-induced glucose intolerance and hyperglycemia. These results suggest that chronic activation of β-cell M3Rs may represent a useful approach to boost insulin output in the long-term treatment of type 2 diabetes.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Antinociceptive mechanism of the spirocyclopiperazinium compound LXM-10 in mice and rats

Drugs typically used to treat pain are limited by their undesirable side effects, which has prompted a search for mechanistically different analgesic agents. We report the antinociception effect of the spirocyclopiperazinium compound LXM-10 via activation of peripheral α7 nicotinic and muscarinic acetylcholine receptors in mice. This effect was attenuated by hexamethonium, atropine methylnitrate, methyllycaconitine citrate, tropicamide, bicuculline, and phaclofen. Competition receptor-binding assays in vitro showed that LXM-10 binds with high affinity α7 nAchR and with low affinity M4 receptors. Our findings show that the antinociception signaling pathway of LXM-10 underlies activation of peripheral α7 nicotinic and possibly of M4 muscarinic receptors, which activate GABA A and GABA B receptors, resulting in antinociceptive effects without obvious side effects.

Modulation of ethanol consumption by genetic and pharmacological manipulation of nicotinic acetylcholine receptors in mice.

Alcohol and nicotine are commonly co-abused. Genetic correlations between responses to these drugs have been reported, providing evidence that common genes underlie the response to alcohol and nicotine. Nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine system are important in mediating nicotine response, and several studies suggest that alcohol may also interact with these nAChRs. The aim of this study was to examine the role of nAChRs containing α7 or β2 subunits in ethanol consumption. A two-bottle choice paradigm was used to determine ethanol consumption in wild-type and nAChR subunit knockout mice. Challenge studies were performed using the α4β2 nAChR partial agonist varenicline. Mice lacking the β2 subunit consumed a similar amount of ethanol compared to their wild-type siblings in an ethanol-drinking paradigm. In contrast, mice lacking the α7 nAChR receptor subunit consumed significantly less ethanol than wild-type mice but consumed comparable amounts of water, saccharin, and quinine. In C57BL/6J mice, varenicline dose-dependently decreased ethanol consumption with a significant effect of 2 mg/kg, without affecting water or saccharin consumption. This effect of varenicline was not reversed in mice lacking either the α7 or β2 subunit, providing evidence that nAChRs containing one of these subunits are not required for this effect of varenicline. This study provides evidence that α7 nAChRs are involved in ethanol consumption and supports the idea that pharmacological manipulation of nAChRs reduces ethanol intake. Additional nAChRs may also be involved in ethanol intake, and there may be functional redundancy in the nicotinic control of alcohol drinking.

Rapsyn interacts with the muscle acetylcholine receptor via α-helical domains in the α, β, and ε subunit intracellular loops

At the developing vertebrate neuromuscular junction, the acetylcholine receptor becomes aggregated at high density in the postsynaptic muscle membrane. Receptor localization is regulated by the motoneuron-derived factor, agrin, and requires an intracellular, scaffolding protein called rapsyn. However, it remains unclear where rapsyn binds on the acetylcholine receptor and how their interaction is regulated. In this study, we identified rapsyn's binding site on the acetylcholine receptor using chimeric constructs where the intracellular domain of CD4 was substituted for the major intracellular loop of each mouse acetylcholine receptor subunit. When expressed in heterologous cells, we found that rapsyn clustered and cytoskeletally anchored CD4-α, β and ε subunit loops but not CD4-δ loop. Rapsyn-mediated clustering and anchoring was highest for β loop, followed by ε and α, suggesting that rapsyn interacts with the loops with different affinities. Moreover, by making deletions within the β subunit intracellular loop, we show that rapsyn interacts with the α-helical region, a secondary structural motif present in the carboxyl terminal portion of the subunit loops. When expressed in muscle cells, rapsyn co-immunoprecipitated together with a CD4-α helical region chimera, independent of agrin signaling. Together, these findings demonstrate that rapsyn interacts with the acetylcholine receptor via an α-helical structural motif conserved between the α, β and ε subunits. Binding at this site likely mediates the critical rapsyn interaction involved in localizing the acetylcholine receptor at the neuromuscular junction.

Acetylcholine receptor and behavioral deficits in mice lacking apolipoprotein E

Apolipoprotein E (apoE) is involved in the risk to develop sporadic Alzheimer's disease (AD). Since impaired central acetylcholine (ACh) function is a hallmark of AD, apoE may influence ACh function by modulating muscarinic ACh receptors (mAChRs). To test this hypothesis, mAChR binding was measured in mice lacking apoE and wild type C57BL/6J mice. Mice were also tested on the pre-pulse inhibition, delay eyeblink classical conditioning, and 5-choice serial reaction time tasks (5-SRTT), which are all modulated by ACh transmission. Mice were also given scopolamine to challenge central mAChR function. Compared to wild type mice, mice lacking apoE had reduced number of cortical and hippocampal mAChRs. Scopolamine had a small effect on delay eyeblink classical conditioning in wild type mice but a large effect in mice lacking apoE. Mice lacking apoE were also unable to acquire performance on the 5-SRTT. These results support a role for apoE in ACh function and suggest that modulation of cortical and hippocampal mAChRs might contribute to genotype differences in scopolamine sensitivity and task acquisition. Impaired apoE functioning may result in cholinergic deficits that contribute to the cognitive impairments seen in AD.

Nicotine Leads to Improvements in Behavioral Impairment and an Increase in the Nicotine Acetylcholine Receptor in Transgenic Mice

Nicotine is the principal psychoactive ingredient in cigarette smoke, and has been associated with health problems in humans. However, the pure form of nicotine may prove to be a valuable pharmaceutical agent for the treatment of AD. However, the beneficial effects of nicotine remain a matter of much controversy. In order to clarify this issue, 12-month-old transgenic mice, expressing neuron-specific enolase (NSE)-controlled APPsw, were treated with low, middle, and high doses of nicotine for 6 months. Herein, we have concluded that the nicotine-treated groups evidenced improvements in behavior and increases in the nicotine acetylcholine receptor, nAchRalpha7. These findings provide experimental evidence that nicotine effects an improvement in impaired memory, and that this improvement is associated with an increase in nAchRalpha7. Thus, nicotine may prove a good preventative or therapeutic modality for AD patients.

Induction of antibodies to particular sites of the α7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).

Mutation of single murine acetylcholine receptor subunits reveals differential contribution of P121 to acetylcholine binding and channel opening

The nicotinic acetylcholine receptor (AChR) is a heteropentameric, ligand-gated ion channel at the neuromuscular junction, where it is responsible for signal transduction between the motorneuron and the muscle. Point mutations in the subunits of the receptor change the channel's electrophysiological properties and underlie inherited forms of muscle weakness, the congenital myasthenic syndromes. One point mutation (P121L) has been identified in the epsilon-subunit of patients suffering from the fast-channel congenital myasthenic syndrome, which is evoked by reduced AChR openings. We introduced the P121L mutation into all murine AChR subunits and performed electrophysiological studies in Xenopus laevis oocytes. The P121L mutation in the epsilon-subunit of the adult mouse AChR affected ligand binding and channel gating in a manner similar to that described for human AChR. At equivalent positions in the alpha- and beta-subunits, the mutation caused only minor electrophysiological changes. Mutation of the delta-subunit had similar, but less pronounced functional consequences compared to epsilonP121L, reflecting the asymmetry of the acetylcholine binding sites and the dominant effect of the alpha-epsilon site on channel opening.

Cholinergically stimulated gastric acid secretion is mediated by M3 and M5 but not M1 muscarinic acetylcholine receptors in mice

Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine; nonetheless, the precise role of each receptor subtype (M(1)-M(5)) remains unclear. This study examined the involvement of M(1), M(3), and M(5) receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. M(3) KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, M(1) KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulated by carbachol, histamine, and gastrin. Pirenzepine, known as an M(1)-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in M(1) KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of M(1)-receptor antagonism. Notably, M(5) KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed M(5)-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by M(3) (on parietal cells) and M(5) receptors (conceivably in the submucosal plexus), but not M(1) receptors.

Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor

The effects of metabolic inhibition of cholesterol biosynthesis on the trafficking of the nicotinic acetylcholine receptor (AChR) to the cell membrane were studied in living CHO-K1/A5, a Chinese hamster ovary clonal line that heterologously expresses adult α 2βδε mouse AChR. To this end, we submitted CHO-K1/A5 cells to long-term cholesterol deprivation, elicited by Mevinolin, a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase and applied a combination of biochemical, pharmacological and fluorescence microscopy techniques to follow the fate of the AChR. When CHO-K1/A5 cells were grown for 48 h in lipid-deficient medium supplemented with 0.5 μM Mevinolin, total cholesterol was significantly reduced (40%). Concomitantly, the maximum number of binding sites ( B max) of the cell-surface AChR for the competitive antagonist α-bungarotoxin was reduced from 647±30 to 352±34 fmol/mg protein, i.e. by 46%. The apparent dissociation constant ( Kd app) for α-bungarotoxin of the AChRs remaining at the cell surface was not modified by cholesterol depletion. Similarly, the half-concentration inhibiting the specific binding of the radioligand (IC 50) for another competitive antagonist, d-tubocurarine, did not differ from that in control cells. The decrease in cell-surface AChR was paralleled by an increase in intracellular AChR levels, which rose from 44±2.1% in control cells to 74±3.3% in Mevinolin-treated cells. When analyzed by wide-field fluorescence microscopy, the fluorescence signal arising from α-bungarotoxin labeled cell-surface AChRs was reduced by approximately 70% in Mevinolin-treated cells. The distribution of intracellular AChR also changed: Alexa 594-α-bungarotoxin-labeled AChR exhibited a highly compartmentalized pattern, concentrating at the perinuclear and Golgi-like regions. Temperature-arrest of protein trafficking magnified this effect, emphasizing the Golgi localization of the AChR. Colocalization studies using the transiently expressed fluorescent trans-Golgi/ trans-Golgi network marker pEYFP/human β1,4-galactosyltransferase and the trans-Golgi network marker syntaxin 6 provided additional support for the Golgi localization of intracellular AChRs. The low AChR cell-surface expression and the increase in intracellular AChR pools in cholesterol-depleted cells raise the possibility that cholesterol participates in the trafficking of the receptor protein to the plasmalemma and its stability at this surface location.

Expression of human-Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG).

The nicotinic AChR, a pentamer composed of alpha2betagamma(or epsilon)delta subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the alpha-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse-Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the alpha-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human alpha+ Torpedo betagammadelta AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the alpha-subunit. Interestingly, the anti-alpha-subunit antibodies predominated in low titre (0.6-7.4 nM) but not in high titre (10-386 nM) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-alpha-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the alpha-subunit.

Experimental autoimmune myasthenia gravis in mice expressing human immunoglobulin loci

Antibodies (Abs) specifically directed against the muscular acetylcholine receptor (AChR) mediate the pathogenesis of myasthenia gravis (MG). The animal model experimental autoimmune MG (EAMG) can be induced by passive transfer or by active immunization of anti-AChR Abs. We report a new EAMG mouse model that generates human anti-AChR Abs upon immunization with Torpedo AChR (tAChR). Mice transgenic for human μ, γ1, and κ germ line genes (HuMAb-Mice) were immunized with tAChR. Serum titers of anti-tAChR Abs were in the nanomolar range, and anti-rodent AChR Abs were in picomolar range. Some HuMAb-Mice had signs of muscle weakness, clearly indicating their susceptibility to EAMG. Human Ab–mouse AChR complexes were found at the neuromuscular junction, while AChR loss was up to 65%. Spleen and lymph nodes were used for producing hybridomas. Of the anti-tAChR monoclonal Ab-producing hybridomas, 2% had cross-reactivity with rodent AChR and none with human AChR. Immunization with a fusion protein, Trx-Hα1-210, displaying the human main immunogenic region did not result in EAMG or the generation of human anti-human AChR monoclonal Abs. These experiments show that the HuMAb-Mouse represents a suitable model to generate and study the effects of human anti-AChR Abs in vivo.

Genetically dystrophic mdx/mdx mice exhibit decreased response to nicotine in passive avoidance.

mdx mice are considered as a genetic homologous of human Duchenne muscular dystrophy. Recent evidence demonstrates that in mouse sympathetic ganglion dystrophin is involved in the stabilization of nicotinic acetylcholine receptor clusters. The purpose of this study was to verify possible effects of dystrophin alterations at the central level. This was assessed by evaluating the response to nicotine administration in mdx and wild-type mice. Thus the effects of post-training nicotine administrations (0.1, 0.25 and 0.5 mg/kg) were tested in mice subjected to a passive avoidance memory task, that measures the ability of mice to remember on test day a shock received 24 h before. Nicotine enhanced memory in wild-type as well as in mdx mice. However, the doses needed to increase memory in mdx were higher than in wild-type. These results are discussed in terms of possible functional changes in central nicotinic acetylcholine receptor in mdx mice.

Mapping myasthenia gravis-associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice.

Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8xDQ6 and DQ8xDR3 F(1) transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (alpha320-337, alpha304-322, and alpha419-437), of which the response to alpha320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR- and alpha320-337 peptide-specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG(2b), and IgG(2c) Ab's and reduced IL-2 and IFN-gamma secretion by H-AChR- and peptide alpha320-337-specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence alpha320-337 as a dominant trait.

Mapping myasthenia gravis–associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice

Mapping myasthenia gravis–associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice

Intradermal Cholinergic Agonists Induce Itch-Associated Response via M3 Muscarinic Acetylcholine Receptors in Mice

In the present study, we examined whether cholinergic agonists would elicit an itch-associated response in mice. When mice were given an intradermal injection of carbachol (1-10 nmol) or bethanechol (0.3-100 nmol) into the rostral back, they showed the dose-dependent increase of scratching. Nicotine (1-10 nmol) showed no effect. Pretreatment with naloxone, but not with terfenadine, significantly suppressed the carbachol-induced scratching. When intradermally co-injected with carbachol, atropine and 4-DAMP but neither methoctramine nor pancuronium significantly inhibited the carbachol-induced scratching. Muscarinic agonists are suggested to produce itch through activation of M3 muscarinic receptors in the skin.