Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus methods
Aim. Evidence-backed execution summary for Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus methods from Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus.
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mouse
Subject model for the experiment.
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Cultures of dissociated DRG neurons
reagent used in the protocol.
- Use
- DRG from all spinal levels of 4-week old mice or rats were collected in cold DH10 medium and treated with enzyme solution at 37°C. Following trituration and centrifugation, cells were resuspended in DH10, plated on glass cover slips coated with poly-D-lysine and laminin, cultured in an incubator at 37°C an...
Cultures of dissociated DRG neurons
reagent used in the protocol.
- Use
- Electroporation of dissociated adult DRG neurons with Mrgpr-expression constructs was carried out using Mouse Neuron Nucleofector Kit (Amaxa Biosystems) following the manufacturer's instructions. Electroporated neurons were plated and cultured as described above.
Mrgpr-clusterΔ -/- mice exhibit severe reduction in CQ-induced scratching
reagent used in the protocol.
- Use
- In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ -/- and WT mice in the total number of scratching bouts induced by histamine over a period of 30 min ( ). Consistent with this r...
Mrgpr-clusterΔ -/- mice exhibit severe reduction in CQ-induced scratching
reagent used in the protocol.
- Use
- Previous studies have indicated that CQ can cause mast cell degranulation (; ). To determine if this effect on mast cells contributes to CQ-evoked scratching behavior, we repeated the experiment on SASH mice, which lack mast cells due to a chromosomal inversion in the regulatory element of the Kit gene ( ). As comp...
CQ directly excites DRG neurons in an Mrgpr-dependent manner
reagent used in the protocol.
- Use
- Since CQ-induced itch is not considered an allergic reaction, we hypothesized given our results that this type of itch results from a direct activation of DRG neurons by the drug. If so, then the behavioral deficit seen in mutant mice would be attributed to a loss of CQ responsiveness in primary sensory neurons. Ind...
CQ directly excites DRG neurons in an Mrgpr-dependent manner
reagent used in the protocol.
- Use
- We performed additional experiments to further characterize [Ca 2+ ] i increases in WT neurons. Sequential application of CQ caused a ~20% reduction of calcium responses. In addition, extracellular Ca 2+ was necessary for the CQ-induced increase in [Ca 2+ ] i since the CQ effect was almost completely blocked in Ca 2...
CQ directly excites DRG neurons in an Mrgpr-dependent manner
reagent used in the protocol.
- Use
- We also examined whether CQ can directly induce action potentials (APs) in dissociated DRG neurons, using whole-cell patch clamp recording. In WT DRG, all CQ-sensitive neurons (identified by calcium imaging) displayed a train of APs upon subsequent CQ treatment ( ). In contrast, all neurons that fail to show a calci...
CQ specifically activates mouse MrgprA3 and human MrgprX1
reagent used in the protocol.
- Use
- The Mrgpr-clusterΔ -/- behavioral and cellular loss-of-function phenotypes strongly suggest that Mrgprs function as cell surface receptors for CQ. To test this possibility directly, we examined whether Mrgprs have the ability to confer sensitivity to CQ on heterologous cells. We cloned each of the t...
EXPERIMENTAL PROCEDURES
To delete a cluster of Mrgpr genes in the mouse germline, we constructed two replacement vectors for MrgprA1 and MrgprB4, which reside on either end of the Mrgpr cluster. The entire open reading frames (ORFs) of both MrgprA1 and MrgprB4 are encoded by a single exon. The arms of the MrgprA1 and MrgprB4 targeting con...
- Use
- To delete a cluster of Mrgpr genes in the mouse germline, we constructed two replacement vectors for MrgprA1 and MrgprB4, which reside on either end of the Mrgpr cluster. The entire open reading frames (ORFs) of both MrgprA1 and MrgprB4 are encoded by a single exon. The arms of the MrgprA1 and MrgprB4 targeting con...
Whole-cell current-clamp recordings of cultured DRG neurons
Neurons plated on cover slips were transferred into a chamber with the extracellular solution. Patch pipettes had resistances of 2-4 MΩ. In current clamp recordings, action potential measurements were performed with an Axon 700B amplifier and the pCLAMP 9.2 software package (Axon Instruments). Neurons we...
- Use
- Neurons plated on cover slips were transferred into a chamber with the extracellular solution. Patch pipettes had resistances of 2-4 MΩ. In current clamp recordings, action potential measurements were performed with an Axon 700B amplifier and the pCLAMP 9.2 software package (Axon Instruments). Neurons we...
Figures
Targeted deletion of a cluster of Mrgpr genes. ( A ) Top horizontal line represents Mrgpr gene cluster on WT mouse chromosome 7. The distance between MrgprA1 and MrgprB4 is 845 kilobases, which contains 12 intact Mrgprs (each represented by a black bar with its name on top). Targeting constructs containing loxP site...
- Use
- Targeted deletion of a cluster of Mrgpr genes. ( A ) Top horizontal line represents Mrgpr gene cluster on WT mouse chromosome 7. The distance between MrgprA1 and MrgprB4 is 845 kilobases, which contains 12 intact Mrgprs (each represented by a black bar with its name on top). Targeting constructs containing loxP site...
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Cultures of dissociated DRG neurons
DRG from all spinal levels of 4-week old mice or rats were collected in cold DH10 medium and treated with enzyme solution at 37°C. Following trituration and centrifugation, cells were resuspended in DH10, plated on glass cover slips coated with poly-D-lysine and laminin, cultured in an incubator at 37°C and used within 24 hours. Details are available (see ).
Mrgpr-clusterΔ -/- mice exhibit severe reduction in CQ-induced scratching
In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ -/- and WT mice in the total number of scratching bouts induced by histamine over a period of 30 min ( ). Consistent with this result, WT and mutant mice also showed similar responses to compound 48/80, a drug that elicits mast cell degranulation and induces histamine-dependent itch (; ) ( ). These results suggest that Mrgprs are not involved in histamine-dependent itch.
Mrgpr-clusterΔ -/- mice exhibit severe reduction in CQ-induced scratching
Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection. Typically, the first bout was observed within 1 to 2 min after injection, and scratching peaked within 15 min in WT mice. In contrast, Mrgpr-clusterΔ -/- mice showed a delayed occurrence of the first scratching behavior (WT 57.8 ± 25.4 sec versus KO 280.5 ± 35.0 sec; P=0.0004). The total number of scratching bouts induced by CQ were 278 ± 21 in WT mice and 104 ± 13 in Mrgpr-clusterΔ -/- mice ( ). Interestingly, injection of a CQ precursor, quinoline, did not evoke any scratching behavior (see below for quinoline's structure and inability of activating Mrgprs) in both WT and mutant mice ( ). However, immediately after quinoline treatment, CQ injection...
CQ directly excites DRG neurons in an Mrgpr-dependent manner
Since CQ-induced itch is not considered an allergic reaction, we hypothesized given our results that this type of itch results from a direct activation of DRG neurons by the drug. If so, then the behavioral deficit seen in mutant mice would be attributed to a loss of CQ responsiveness in primary sensory neurons. Indeed, 1 mM CQ treatment of cultured DRG neurons evoked a robust intracellular calcium ([Ca 2+ ] i ) increase in ~4-5% of the cells from WT mice. In contrast, none of the neurons in cultures derived from Mrgpr-deficient mice exhibited any significant response to CQ ( ). These data indicate that the CQ-evoked [Ca 2+ ] i increases seen in WT DRG cultures reflect specific activation of a subset of cells, and that this activation is Mrgpr-dependent. In contrast, the percentage of DRG neurons responding to histamine was identical between WT and mutant cultures, consistent wi...
CQ specifically activates mouse MrgprA3 and human MrgprX1
In order to determine the lowest concentrations of CQ capable of activating MrgprA3 and X1, we performed dose-response experiments in HEK293 cells. These experiments indicated that the receptors could be activated by the drug at micromolar concentrations with the mouse receptor showing 10-fold higher sensitivity than the human receptor ( ). EC 50 s for MrgprA3 and MrgprX1 are 27.55 ± 2.03 and 297.68 ±2.10 µM, respectively (see Discussion regarding the low potency of CQ on MrgprX1). Besides CQ, we also determined the sensitivities of these receptors to other structurally related compounds (i.e. quinoline, quinine and serotonin) ( ). Quinoline is used as an intermediate in the production of various compounds including CQ. Despite the presence of a bicyclic structure, quinoline completely failed to activate MrgprA3 and MrgprX1 suggesting that the side chain in CQ is also n...
MrgprA3 and MrgprX1 rescue the phenotypes of Mrgpr-deficient neurons
We next asked whether MrgprA3 or MrgprX1 can rescue the phenotypes of DRG neurons from Mrgpr-clusterΔ -/- mice. To answer this question, we electroporated the Mrgpr expression constructs used in the heterologous studies into dissociated adult DRG neurons from these mice. After 24 hour in culture, expression and membrane localization of the transfected Mrgprs in the mutant neurons could be readily visualized by GFP ( ). Strikingly, all MrgprA3-expressing mutant neurons generated numerous APs in response to CQ treatment ( ) whereas neighboring GFP-negative neurons remained silent (n=6, not shown). The number of APs generated in the GFP-positive neurons was comparable to that produced by CQ treatment of WT DRG neurons, indicating a nearly complete rescue by MrgprA3. Similar results were obtained for Mrgpr-deficient neurons electroporated with MrgprX1 ( ). In contrast, fe...
Behavioral studies
All behavioral tests were performed with an experimenter blind to genotype. The mice were 2 to 3 month-old males (20-30 g) that had been backcrossed to C57Bl/6 mice for at least ten generations. Rat studies were done using four week old CD® animals (Charles River Laboratories). All experiments were performed under the protocol approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. Pruritic compounds (i.e. histamine, compound 48/80, and CQ) were subcutaneously injected into the nape of the neck after acclimatization and scratching behavior was observed for 30 min. A bout of scratching was defined as continuous scratch movements with hind paws directed at the area around the injection site. Scratching behavior was quantified by recording the number of scratching bouts at 5 min intervals over the 30 min observation period. Details for oth...
Calcium imaging
Neurons or HEK293 cells were loaded with fura 2-acetomethoxy ester (Molecular Probes) for 30 min in the dark at room temperature or 45 min at 37°C, respectively. After washing, cells were imaged at 340 and 380 nm excitation to detect intracellular free calcium. Calcium imaging assays were performed with an experimenter blind to genotype. Each experiment was done at least three times and at least 100 cells (neurons or HEK293 cells) were analyzed each time.
Measurement outputs
What raw and processed outputs should exist?
Electroporation of dissociated adult DRG neurons with Mrgpr-expression constructs was carried out using Mouse Neuron Nucleofector Kit (Amaxa Biosystems) following the manufactur...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection....
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Previous studies have indicated that CQ can cause mast cell degranulation (; ). To determine if this effect on mast cells contributes to CQ-evoked scratching behavior, we repea...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The Mrgpr-clusterΔ -/- behavioral and cellular loss-of-function phenotypes strongly suggest that Mrgprs function as cell surface receptors for CQ. To test this...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Activation of small-diameter sensory neurons in DRG can generate different types of somatosensation including pain and itch with specific and distinct behavioral responses.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Electroporation of dissociated adult DRG neurons with Mrgpr-expression constructs was carried out using Mouse Neuron Nucleofector Kit (Amaxa Biosystems) following the manufactur...; Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection....; Previous studies have indicated that CQ can cause mast cell degranulation (; ). To determine if this effect on mast cells contributes to CQ-evoked scratching behavior, we repea...; The Mrgpr-clusterΔ -/- behavioral and cellular loss-of-function phenotypes strongly suggest that Mrgprs function as cell surface receptors for CQ. To test this....
from paperStatistical comparison
Activation of small-diameter sensory neurons in DRG can generate different types of somatosensation including pain and itch with specific and distinct behavioral responses. For...; In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ...; Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection....; Previous studies have indicated that CQ can cause mast cell degranulation (; ). To determine if this effect on mast cells contributes to CQ-evoked scratching behavior, we repea...
from paperReporting output
Report representative outputs alongside summary comparisons for Electroporation of dissociated adult DRG neurons with Mrgpr-expression constructs was carried out using Mouse Neuron Nucleofector Kit (Amaxa Biosystems) following the manufactur..., Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection...., Previous studies have indicated that CQ can cause mast cell degranulation (; ). To determine if this effect on mast cells contributes to CQ-evoked scratching behavior, we repea..., The Mrgpr-clusterΔ -/- behavioral and cellular loss-of-function phenotypes strongly suggest that Mrgprs function as cell surface receptors for CQ. To test this....
inferred from protocolStructured statistical methods
Activation of small-diameter sensory neurons in DRG can generate different types of somatosensation including pain and itch with specific and distinct behavioral responses. For...; In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ...; Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection....; Previous studies have indicated that CQ can cause mast cell degranulation (; ). To determine if this effect on mast cells contributes to CQ-evoked scratching behavior, we repea...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
DRG from all spinal levels of 4-week old mice or rats were collected in cold DH10 medium and treated with enzyme solution at 37°C. Following trituration and centrifugation, cells were resuspended in DH10, plated on glass cover slips coated with poly-D-lysine and laminin, cultured in an incubator at 37°C and used within 24 hours. Details are available (see ).
In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ -/- and WT mice in the total number of scratching bouts induced by histamine over a period of 30 min ( ). Consistent with this result, WT and mutant mice also showed similar responses to compound 48/80, a drug that elicits mast cell degranulation and induces histamine-dependent itch (; ) ( ). These results suggest that Mrgprs are not involved in histamine-dependent itch.
Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection. Typically, the first bout was observed within 1 to 2 min after injection, and scratching peaked within 15 min in WT mice. In contrast, Mrgpr-clusterΔ -/- mice showed a delayed occurrence of the first scratching behavior (WT 57.8 ± 25.4 sec versus KO 280.5 ± 35.0 sec; P=0.0004). The total number of scratching bouts induced by CQ were 278 ± 21 in WT mice and 104 ± 13 in Mrgpr-clusterΔ -/- mice ( ). Interestingly, injection of a CQ precursor, quinoline, did not evoke any scratching behavior (see below for quinoline's structure and inability of activating Mrgprs) in both WT and mutant mice ( ). However, immediately after quinoline treatment, CQ injection at the same location induced robust scratching behavior in WT mice; and the number of scratches induced by this treatment was again severely reduced in Mrgpr-clusterΔ -/- mice ( ). These results suggest that CQ-induced itch, but not histaminergic itch, is affected in the cluster del...
Since CQ-induced itch is not considered an allergic reaction, we hypothesized given our results that this type of itch results from a direct activation of DRG neurons by the drug. If so, then the behavioral deficit seen in mutant mice would be attributed to a loss of CQ responsiveness in primary sensory neurons. Indeed, 1 mM CQ treatment of cultured DRG neurons evoked a robust intracellular calcium ([Ca 2+ ] i ) increase in ~4-5% of the cells from WT mice. In contrast, none of the neurons in cultures derived from Mrgpr-deficient mice exhibited any significant response to CQ ( ). These data indicate that the CQ-evoked [Ca 2+ ] i increases seen in WT DRG cultures reflect specific activation of a subset of cells, and that this activation is Mrgpr-dependent. In contrast, the percentage of DRG neurons responding to histamine was identical between WT and mutant cultures, consistent with the behavioral data and providing further evidence that histamine-induced itch is unaffected in Mrgpr-clusterΔ -/- mice ( ).
In order to determine the lowest concentrations of CQ capable of activating MrgprA3 and X1, we performed dose-response experiments in HEK293 cells. These experiments indicated that the receptors could be activated by the drug at micromolar concentrations with the mouse receptor showing 10-fold higher sensitivity than the human receptor ( ). EC 50 s for MrgprA3 and MrgprX1 are 27.55 ± 2.03 and 297.68 ±2.10 µM, respectively (see Discussion regarding the low potency of CQ on MrgprX1). Besides CQ, we also determined the sensitivities of these receptors to other structurally related compounds (i.e. quinoline, quinine and serotonin) ( ). Quinoline is used as an intermediate in the production of various compounds including CQ. Despite the presence of a bicyclic structure, quinoline completely failed to activate MrgprA3 and MrgprX1 suggesting that the side chain in CQ is also necessary for activation ( ). Consistently, quinoline does not induce any scratching behavior in mice ( ). Serotonin also has a bicyclic structure. But unlike CQ or quinoline, its bicyclic structure consists of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring ( )....
We next asked whether MrgprA3 or MrgprX1 can rescue the phenotypes of DRG neurons from Mrgpr-clusterΔ -/- mice. To answer this question, we electroporated the Mrgpr expression constructs used in the heterologous studies into dissociated adult DRG neurons from these mice. After 24 hour in culture, expression and membrane localization of the transfected Mrgprs in the mutant neurons could be readily visualized by GFP ( ). Strikingly, all MrgprA3-expressing mutant neurons generated numerous APs in response to CQ treatment ( ) whereas neighboring GFP-negative neurons remained silent (n=6, not shown). The number of APs generated in the GFP-positive neurons was comparable to that produced by CQ treatment of WT DRG neurons, indicating a nearly complete rescue by MrgprA3. Similar results were obtained for Mrgpr-deficient neurons electroporated with MrgprX1 ( ). In contrast, fewer than half of the MrgprA1-electroporated neurons elicited a few APs in response to CQ ( ). Rescue by MrgprA3 and MrgprX1 was also seen using calcium imaging, with an increase in [Ca 2+ ] i induced by CQ ( ). Together these results strongly suggest that mouse MrgprA3 and human MrgprX1 are the majo...
All behavioral tests were performed with an experimenter blind to genotype. The mice were 2 to 3 month-old males (20-30 g) that had been backcrossed to C57Bl/6 mice for at least ten generations. Rat studies were done using four week old CD® animals (Charles River Laboratories). All experiments were performed under the protocol approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. Pruritic compounds (i.e. histamine, compound 48/80, and CQ) were subcutaneously injected into the nape of the neck after acclimatization and scratching behavior was observed for 30 min. A bout of scratching was defined as continuous scratch movements with hind paws directed at the area around the injection site. Scratching behavior was quantified by recording the number of scratching bouts at 5 min intervals over the 30 min observation period. Details for other behavior assays are available in the.
Neurons or HEK293 cells were loaded with fura 2-acetomethoxy ester (Molecular Probes) for 30 min in the dark at room temperature or 45 min at 37°C, respectively. After washing, cells were imaged at 340 and 380 nm excitation to detect intracellular free calcium. Calcium imaging assays were performed with an experimenter blind to genotype. Each experiment was done at least three times and at least 100 cells (neurons or HEK293 cells) were analyzed each time.
Machine-readable layer
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"description": "Evidence-backed execution summary for Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus methods from Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus.",
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"text": "In addition to pain, we evaluated chemically induced itch responses in Mrgpr-clusterΔ -/- mice. No significant difference was found between Mrgpr-clusterΔ -/- and WT mice in the total number of scratching bouts induced by histamine over a period of 30 min ( ). Consistent with this result, WT and mutant mice also showed similar responses to compound 48/80, a drug that elicits mast cell degranulation and induces histamine-dependent itch (; ) ( ). These results suggest that Mrgprs are not involved in histamine-dependent itch."
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"text": "Strikingly, itch induced by CQ was strongly reduced in Mrgpr-clusterΔ -/- mice. shows the time course of scratching bouts at 5 min intervals after CQ injection. Typically, the first bout was observed within 1 to 2 min after injection, and scratching peaked within 15 min in WT mice. In contrast, Mrgpr-clusterΔ -/- mice showed a delayed occurrence of the first scratching behavior (WT 57.8 ± 25.4 sec versus KO 280.5 ± 35.0 sec; P=0.0004). The total number of scratching bouts induced by CQ were 278 ± 21 in WT mice and 104 ± 13 in Mrgpr-clusterΔ -/- mice ( ). Interestingly, injection of a CQ precursor, quinoline, did not evoke any scratching behavior (see below for quinoline's structure and inability of activating Mrgprs) in both WT and mutant mice ( ). However, immediately after quinoline treatment, CQ injection..."
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"text": "Since CQ-induced itch is not considered an allergic reaction, we hypothesized given our results that this type of itch results from a direct activation of DRG neurons by the drug. If so, then the behavioral deficit seen in mutant mice would be attributed to a loss of CQ responsiveness in primary sensory neurons. Indeed, 1 mM CQ treatment of cultured DRG neurons evoked a robust intracellular calcium ([Ca 2+ ] i ) increase in ~4-5% of the cells from WT mice. In contrast, none of the neurons in cultures derived from Mrgpr-deficient mice exhibited any significant response to CQ ( ). These data indicate that the CQ-evoked [Ca 2+ ] i increases seen in WT DRG cultures reflect specific activation of a subset of cells, and that this activation is Mrgpr-dependent. In contrast, the percentage of DRG neurons responding to histamine was identical between WT and mutant cultures, consistent wi..."
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"text": "In order to determine the lowest concentrations of CQ capable of activating MrgprA3 and X1, we performed dose-response experiments in HEK293 cells. These experiments indicated that the receptors could be activated by the drug at micromolar concentrations with the mouse receptor showing 10-fold higher sensitivity than the human receptor ( ). EC 50 s for MrgprA3 and MrgprX1 are 27.55 ± 2.03 and 297.68 ±2.10 µM, respectively (see Discussion regarding the low potency of CQ on MrgprX1). Besides CQ, we also determined the sensitivities of these receptors to other structurally related compounds (i.e. quinoline, quinine and serotonin) ( ). Quinoline is used as an intermediate in the production of various compounds including CQ. Despite the presence of a bicyclic structure, quinoline completely failed to activate MrgprA3 and MrgprX1 suggesting that the side chain in CQ is also n..."
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"text": "We next asked whether MrgprA3 or MrgprX1 can rescue the phenotypes of DRG neurons from Mrgpr-clusterΔ -/- mice. To answer this question, we electroporated the Mrgpr expression constructs used in the heterologous studies into dissociated adult DRG neurons from these mice. After 24 hour in culture, expression and membrane localization of the transfected Mrgprs in the mutant neurons could be readily visualized by GFP ( ). Strikingly, all MrgprA3-expressing mutant neurons generated numerous APs in response to CQ treatment ( ) whereas neighboring GFP-negative neurons remained silent (n=6, not shown). The number of APs generated in the GFP-positive neurons was comparable to that produced by CQ treatment of WT DRG neurons, indicating a nearly complete rescue by MrgprA3. Similar results were obtained for Mrgpr-deficient neurons electroporated with MrgprX1 ( ). In contrast, fe..."
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