The DREADD agonist clozapine N -oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Materials and Methods
Ten adult male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA, USA) weighing approximately 250-450 g over the duration of the study served as subjects for drug discrimination experiments. A separate cohort of eight adult male Sprague-Dawley rats (Charles River Laboratories Inc.) served as subjects for the pharmacokinetic studies. Rats were individually housed in a climate-controlled room under a reverse 12-h light/dark cycle (lights on 2000 to 0800). The rats serving in drug discrimination experiments were maintained at ~90% free-feeding weight by providing 16-18 g of standard rodent chow daily approximately 30-60 min following training/test sessions, while the eight rats employed in pharmacokinetic studies were provided food ad libitum in the home cage throughout the duration of experiments. Water was available ad libitum to a...
Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice
Plasma levels of clozapine, CNO, and the active clozapine metabolite N -desmethylclozapine (NDMC) were quantified in rats and mice following administration of the 1.25 mg/kg training dose of clozapine or 10.0 mg/kg CNO, a dose that both has been used to activate DREADDs and produced moderate (rats) and high (mice) levels of substitution for clozapine in Experiment 1 (Fig. ).
Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice
Administration of 1.25 mg/kg clozapine in rats (Fig. ) resulted in 10.63 ± 1.99 ng/ml clozapine in plasma 30 min post injection, which decreased only slightly to 8.52 ± 2.12 ng/ml by the 60 min time point. Levels of the two primary metabolites for clozapine (CNO and NDMC) were below limits of detection 30 min post injection, but emerged at very low quantities (<0.5 ng/ml) at 60 min. The ratios of CNO to clozapine and NDMC to clozapine at 60 min post clozapine injection were 5.4% and 3.8%, respectively. In general, the levels of clozapine detected and the pharmacokinetic profile we observed were very similar to those reported previously in the same rat strain using a nearly-identical dose of clozapine (1.0 mg/kg). Figure 3 CNO is converted to clozapine in standard laboratory rats and mice. Plasma samples...
Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice
Administration of 10.0 mg/kg CNO to rats resulted in a robust rise in plasma CNO levels (peak at 30 min; 3,404.13 ± 596.84 ng/ml), accompanied by the simultaneous emergence of clozapine at levels far higher than those produced by the training dose of 1.25 mg/kg clozapine (peak at 30 min; 256.73 ± 214.56 ng/ml) (Fig. ). The ratio of clozapine to CNO at the 30 min time point in our Sprague-Dawley rats was 7.5%, similar to that reported following 5.0 mg/kg CNO administration in Long-Evans rats (~13%) at the same time point. It was interesting to note that in both studies, the emergence of NDMC increased gradually over a period of time during which levels of clozapine and CNO were decreasing. Furthermore, our calculated NDMC to clozapine ratio at 60 min post CNO administration was 3.6%, which was almost ide...
Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice
While the 10.0 mg/kg CNO dose was selected in this experiment because it engendered maximal average substitution for the clozapine discriminative stimulus, lower doses are more typically employed to activate DREADDs in rats. We therefore quantified levels of CNO, clozapine, and NDMC following administration of 1.0 mg/kg CNO in rats, a dose which is frequently used in DREADD studies and which showed partial-to-full substitution in a small subset of discrimination subjects. Administration of 1.0 mg/kg CNO to rats resulted again in a measurable rise in plasma CNO levels (peak at 30 min; 51.40 ± 7.16 ng/ml) (Fig. ). In contrast to the higher dose of 10.0 mg/kg however, neither clozapine nor NDMC were detectable in plasma above limits of detection. Figure 4 Pharmacokinetic analysis following administration of 1.0 mg/kg CNO i.p. in...
Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice
Administration of the 1.25 mg/kg training dose of clozapine in mice (Fig. ) produced mean clozapine plasma levels of 143.53 ± 32.21 ng/ml 30 min post injection which decreased approximately 2.2-fold to 65.33 ng/ml ± 12.93 by 60 min. NDMC was detectable alongside clozapine and, as was observed in the rat, increased slightly while clozapine levels decreased over time. Interestingly, CNO was not detected following this low-dose clozapine administration, reinforcing previous observations that NDMC is the primary metabolite in this species,. Administration of 10.0 mg/kg CNO to mice resulted in an expected rise in plasma CNO levels (peak at 30 min; 623.7 ± 114.1 ng/ml) which decreased roughly 5.5 fold by 60 min (Fig. ). More important was the detection of high levels of clozapine and NDMC at...
Materials and Methods
Ten adult B6129 inbred mice (male, n = 7; female, n = 3) weighing between 20-30 g (Harlan Laboratories, Indianapolis, IN, USA) served as subjects in drug discrimination experiments. These mice were individually housed in clear plastic cages within a climate-controlled vivarium on a 12 h light/dark cycle (0600/1800 and maintained at 85-90% free-feeding body weights on standard rodent chow which was made available in the home cage ~30 min after daily training/testing for the duration of the study. Water was available ad libitum in home cages. Mice were moved daily (6 to 7 days each week) from the vivarium to the laboratory where testing occurred.
Drug discrimination procedure
Rats and mice were trained to discriminate 1.25 mg/kg clozapine from its vehicle using a two-lever, food-reinforced drug discrimination procedure based on methods published previously, - and described in detail in the Supplementary Materials and Methods. Briefly, each animal was assigned one lever as the "clozapine-appropriate lever", and the other lever as the "vehicle-appropriate lever." When the subject was injected with clozapine, only responses on the "clozapine-appropriate lever" were reinforced, whereas only responses on the "vehicle-appropriate lever" were reinforced following injection of vehicle. Animals received only one injection per day. The 1.25 mg/kg dose of clozapine was selected as the training dose in these studies because this is the lowest dose of clozapine that has been reported to function as...
Measurement outputs
What raw and processed outputs should exist?
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
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (8)
Ten adult male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA, USA) weighing approximately 250-450 g over the duration of the study served as subjects for drug discrimination experiments. A separate cohort of eight adult male Sprague-Dawley rats (Charles River Laboratories Inc.) served as subjects for the pharmacokinetic studies. Rats were individually housed in a climate-controlled room under a reverse 12-h light/dark cycle (lights on 2000 to 0800). The rats serving in drug discrimination experiments were maintained at ~90% free-feeding weight by providing 16-18 g of standard rodent chow daily approximately 30-60 min following training/test sessions, while the eight rats employed in pharmacokinetic studies were provided food ad libitum in the home cage throughout the duration of experiments. Water was available ad libitum to all rats in their home cage. Behavioral experiments were conducted 5-6 days/week in operant chambers located within the vivarium between the hours of 1300 and 1600.
Plasma levels of clozapine, CNO, and the active clozapine metabolite N -desmethylclozapine (NDMC) were quantified in rats and mice following administration of the 1.25 mg/kg training dose of clozapine or 10.0 mg/kg CNO, a dose that both has been used to activate DREADDs and produced moderate (rats) and high (mice) levels of substitution for clozapine in Experiment 1 (Fig. ).
Administration of 1.25 mg/kg clozapine in rats (Fig. ) resulted in 10.63 ± 1.99 ng/ml clozapine in plasma 30 min post injection, which decreased only slightly to 8.52 ± 2.12 ng/ml by the 60 min time point. Levels of the two primary metabolites for clozapine (CNO and NDMC) were below limits of detection 30 min post injection, but emerged at very low quantities (<0.5 ng/ml) at 60 min. The ratios of CNO to clozapine and NDMC to clozapine at 60 min post clozapine injection were 5.4% and 3.8%, respectively. In general, the levels of clozapine detected and the pharmacokinetic profile we observed were very similar to those reported previously in the same rat strain using a nearly-identical dose of clozapine (1.0 mg/kg). Figure 3 CNO is converted to clozapine in standard laboratory rats and mice. Plasma samples were collected 30 min and 60 min after injection of 1.25 mg/kg clozapine or 10.0 mg/kg CNO and analyzed via UPLC-LC-MS/MS for concentrations of clozapine, CNO, and N -desmethylclozapine (NDMC). Data for each analyte are presented as the mean (±SEM) concentration. Mean v...
Administration of 10.0 mg/kg CNO to rats resulted in a robust rise in plasma CNO levels (peak at 30 min; 3,404.13 ± 596.84 ng/ml), accompanied by the simultaneous emergence of clozapine at levels far higher than those produced by the training dose of 1.25 mg/kg clozapine (peak at 30 min; 256.73 ± 214.56 ng/ml) (Fig. ). The ratio of clozapine to CNO at the 30 min time point in our Sprague-Dawley rats was 7.5%, similar to that reported following 5.0 mg/kg CNO administration in Long-Evans rats (~13%) at the same time point. It was interesting to note that in both studies, the emergence of NDMC increased gradually over a period of time during which levels of clozapine and CNO were decreasing. Furthermore, our calculated NDMC to clozapine ratio at 60 min post CNO administration was 3.6%, which was almost identical to the ratio of 3.8% following administration of clozapine. Combined, these results suggest a pharmacokinetic profile in which systemically-administered CNO is rapidly converted to clozapine in the rat, with the subsequent metabolism of clozapine to CNO and NDMC occurring gradually.
While the 10.0 mg/kg CNO dose was selected in this experiment because it engendered maximal average substitution for the clozapine discriminative stimulus, lower doses are more typically employed to activate DREADDs in rats. We therefore quantified levels of CNO, clozapine, and NDMC following administration of 1.0 mg/kg CNO in rats, a dose which is frequently used in DREADD studies and which showed partial-to-full substitution in a small subset of discrimination subjects. Administration of 1.0 mg/kg CNO to rats resulted again in a measurable rise in plasma CNO levels (peak at 30 min; 51.40 ± 7.16 ng/ml) (Fig. ). In contrast to the higher dose of 10.0 mg/kg however, neither clozapine nor NDMC were detectable in plasma above limits of detection. Figure 4 Pharmacokinetic analysis following administration of 1.0 mg/kg CNO i.p. in rats. Plasma samples were collected 30 min and 60 min after injection CNO and analyzed via LC-MS/MS for concentrations of clozapine, CNO, and N -desmethylclozapine (NDMC). Data for each analyte are presented as the mean (±SEM) concentration. A value reported as <0.2 ng/ml indicates...
Administration of the 1.25 mg/kg training dose of clozapine in mice (Fig. ) produced mean clozapine plasma levels of 143.53 ± 32.21 ng/ml 30 min post injection which decreased approximately 2.2-fold to 65.33 ng/ml ± 12.93 by 60 min. NDMC was detectable alongside clozapine and, as was observed in the rat, increased slightly while clozapine levels decreased over time. Interestingly, CNO was not detected following this low-dose clozapine administration, reinforcing previous observations that NDMC is the primary metabolite in this species,. Administration of 10.0 mg/kg CNO to mice resulted in an expected rise in plasma CNO levels (peak at 30 min; 623.7 ± 114.1 ng/ml) which decreased roughly 5.5 fold by 60 min (Fig. ). More important was the detection of high levels of clozapine and NDMC at 30 min post injection (clozapine, 45.9 ng/ml; NDMC, 136.5 ng/ml) and 60 min post injection (clozapine, 44.4 ng/ml; 131.4 ng/ml), demonstrating that CNO unequivocally undergoes significant conversion to clozapine in the mouse. At 30 min post CNO injection, the ratio of clozapine to...
Ten adult B6129 inbred mice (male, n = 7; female, n = 3) weighing between 20-30 g (Harlan Laboratories, Indianapolis, IN, USA) served as subjects in drug discrimination experiments. These mice were individually housed in clear plastic cages within a climate-controlled vivarium on a 12 h light/dark cycle (0600/1800 and maintained at 85-90% free-feeding body weights on standard rodent chow which was made available in the home cage ~30 min after daily training/testing for the duration of the study. Water was available ad libitum in home cages. Mice were moved daily (6 to 7 days each week) from the vivarium to the laboratory where testing occurred.
Rats and mice were trained to discriminate 1.25 mg/kg clozapine from its vehicle using a two-lever, food-reinforced drug discrimination procedure based on methods published previously, - and described in detail in the Supplementary Materials and Methods. Briefly, each animal was assigned one lever as the "clozapine-appropriate lever", and the other lever as the "vehicle-appropriate lever." When the subject was injected with clozapine, only responses on the "clozapine-appropriate lever" were reinforced, whereas only responses on the "vehicle-appropriate lever" were reinforced following injection of vehicle. Animals received only one injection per day. The 1.25 mg/kg dose of clozapine was selected as the training dose in these studies because this is the lowest dose of clozapine that has been reported to function as a discriminative stimulus,.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "The DREADD agonist clozapine N -oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice methods",
"description": "Protocol for The DREADD agonist clozapine N -oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Materials and Methods",
"text": "Ten adult male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA, USA) weighing approximately 250-450 g over the duration of the study served as subjects for drug discrimination experiments. A separate cohort of eight adult male Sprague-Dawley rats (Charles River Laboratories Inc.) served as subjects for the pharmacokinetic studies. Rats were individually housed in a climate-controlled room under a reverse 12-h light/dark cycle (lights on 2000 to 0800). The rats serving in drug discrimination experiments were maintained at ~90% free-feeding weight by providing 16-18 g of standard rodent chow daily approximately 30-60 min following training/test sessions, while the eight rats employed in pharmacokinetic studies were provided food ad libitum in the home cage throughout the duration of experiments. Water was available ad libitum to a..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice",
"text": "Plasma levels of clozapine, CNO, and the active clozapine metabolite N -desmethylclozapine (NDMC) were quantified in rats and mice following administration of the 1.25 mg/kg training dose of clozapine or 10.0 mg/kg CNO, a dose that both has been used to activate DREADDs and produced moderate (rats) and high (mice) levels of substitution for clozapine in Experiment 1 (Fig. )."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice",
"text": "Administration of 1.25 mg/kg clozapine in rats (Fig. ) resulted in 10.63 ± 1.99 ng/ml clozapine in plasma 30 min post injection, which decreased only slightly to 8.52 ± 2.12 ng/ml by the 60 min time point. Levels of the two primary metabolites for clozapine (CNO and NDMC) were below limits of detection 30 min post injection, but emerged at very low quantities (<0.5 ng/ml) at 60 min. The ratios of CNO to clozapine and NDMC to clozapine at 60 min post clozapine injection were 5.4% and 3.8%, respectively. In general, the levels of clozapine detected and the pharmacokinetic profile we observed were very similar to those reported previously in the same rat strain using a nearly-identical dose of clozapine (1.0 mg/kg). Figure 3 CNO is converted to clozapine in standard laboratory rats and mice. Plasma samples..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice",
"text": "Administration of 10.0 mg/kg CNO to rats resulted in a robust rise in plasma CNO levels (peak at 30 min; 3,404.13 ± 596.84 ng/ml), accompanied by the simultaneous emergence of clozapine at levels far higher than those produced by the training dose of 1.25 mg/kg clozapine (peak at 30 min; 256.73 ± 214.56 ng/ml) (Fig. ). The ratio of clozapine to CNO at the 30 min time point in our Sprague-Dawley rats was 7.5%, similar to that reported following 5.0 mg/kg CNO administration in Long-Evans rats (~13%) at the same time point. It was interesting to note that in both studies, the emergence of NDMC increased gradually over a period of time during which levels of clozapine and CNO were decreasing. Furthermore, our calculated NDMC to clozapine ratio at 60 min post CNO administration was 3.6%, which was almost ide..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice",
"text": "While the 10.0 mg/kg CNO dose was selected in this experiment because it engendered maximal average substitution for the clozapine discriminative stimulus, lower doses are more typically employed to activate DREADDs in rats. We therefore quantified levels of CNO, clozapine, and NDMC following administration of 1.0 mg/kg CNO in rats, a dose which is frequently used in DREADD studies and which showed partial-to-full substitution in a small subset of discrimination subjects. Administration of 1.0 mg/kg CNO to rats resulted again in a measurable rise in plasma CNO levels (peak at 30 min; 51.40 ± 7.16 ng/ml) (Fig. ). In contrast to the higher dose of 10.0 mg/kg however, neither clozapine nor NDMC were detectable in plasma above limits of detection. Figure 4 Pharmacokinetic analysis following administration of 1.0 mg/kg CNO i.p. in..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Experiment 2: Pharmacokinetic Analysis of CNO in Rats and Mice",
"text": "Administration of the 1.25 mg/kg training dose of clozapine in mice (Fig. ) produced mean clozapine plasma levels of 143.53 ± 32.21 ng/ml 30 min post injection which decreased approximately 2.2-fold to 65.33 ng/ml ± 12.93 by 60 min. NDMC was detectable alongside clozapine and, as was observed in the rat, increased slightly while clozapine levels decreased over time. Interestingly, CNO was not detected following this low-dose clozapine administration, reinforcing previous observations that NDMC is the primary metabolite in this species,. Administration of 10.0 mg/kg CNO to mice resulted in an expected rise in plasma CNO levels (peak at 30 min; 623.7 ± 114.1 ng/ml) which decreased roughly 5.5 fold by 60 min (Fig. ). More important was the detection of high levels of clozapine and NDMC at..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Materials and Methods",
"text": "Ten adult B6129 inbred mice (male, n = 7; female, n = 3) weighing between 20-30 g (Harlan Laboratories, Indianapolis, IN, USA) served as subjects in drug discrimination experiments. These mice were individually housed in clear plastic cages within a climate-controlled vivarium on a 12 h light/dark cycle (0600/1800 and maintained at 85-90% free-feeding body weights on standard rodent chow which was made available in the home cage ~30 min after daily training/testing for the duration of the study. Water was available ad libitum in home cages. Mice were moved daily (6 to 7 days each week) from the vivarium to the laboratory where testing occurred."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Drug discrimination procedure",
"text": "Rats and mice were trained to discriminate 1.25 mg/kg clozapine from its vehicle using a two-lever, food-reinforced drug discrimination procedure based on methods published previously, - and described in detail in the Supplementary Materials and Methods. Briefly, each animal was assigned one lever as the \"clozapine-appropriate lever\", and the other lever as the \"vehicle-appropriate lever.\" When the subject was injected with clozapine, only responses on the \"clozapine-appropriate lever\" were reinforced, whereas only responses on the \"vehicle-appropriate lever\" were reinforced following injection of vehicle. Animals received only one injection per day. The 1.25 mg/kg dose of clozapine was selected as the training dose in these studies because this is the lowest dose of clozapine that has been reported to function as..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "The DREADD agonist clozapine N -oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice",
"datePublished": "2018",
"author": [
{
"@type": "Person",
"name": "Daniel F. Manvich"
},
{
"@type": "Person",
"name": "Kevin A. Webster"
},
{
"@type": "Person",
"name": "Stephanie L. Foster"
},
{
"@type": "Person",
"name": "Martilias S. Farrell"
},
{
"@type": "Person",
"name": "James C. Ritchie"
},
{
"@type": "Person",
"name": "Joseph H. Porter"
},
{
"@type": "Person",
"name": "David Weinshenker"
}
],
"identifier": "10.1038/s41598-018-22116-z"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "The DREADD agonist clozapine N -oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice methods",
"item": "https://replicatescience.com/experiments/the-dreadd-agonist-clozapine-n-oxide-cno-is-reverse-metabolized-to-clozapine-and-produces-clozapine-like-interoceptive-stimulus-effects-in-rats-and-mice-methods-daniel-f-manvich-pm/the-dreadd-agonist-clozapine-n-oxide-cno-is-reverse-metabolized-to-clozapine-and-produces-clozapine--mlph2ri6"
}
]
}
]