Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments methods
Aim. Evidence-backed execution summary for Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments methods from Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and Methods
reagent used in the protocol.
- Use
- CNO was supplied by the National Institutes of Health Drug Supply Program, and was dissolved in DMSO then diluted to a final concentration of 1, 2, or 5 mg/ml CNO in 0.5% DMSO in saline solution. Control injections were 0.5% DMSO in saline solution. Of the 22 articles that we found that used systemic administration...
Materials and Methods
reagent used in the protocol.
- Use
- Phencyclidine (PCP; Sigma-Aldrich), d -amphetamine (Sigma-Aldrich), and scopolamine (Tocris Bioscience) were dissolved in 0.9% saline solution, and injected at 1 ml/kg.
CNO-alone studies
reagent used in the protocol.
- Use
- To assess the effects of CNO on startle and PPI, rats were injected with CNO (1 mg/kg, i.p.) or 0.5% DMSO in saline solution 20 min before being placed in the PPI chambers. This dose was chosen based on the literature (; ) and consultation with investigators who had experience using DREADDs.
HPLC studies
reagent used in the protocol.
- Use
- Clozapine (assay 98.9%) was obtained from MP Biomedicals. N -desmethylclozapine (assay >99%) and doxipine HCL internal standard (assay >99%) were obtained from Tocris Bioscience. For these studies, only CNO 5 mg/kg was tested because it was efficacious in the voltammetry and locomotor studies. In addition, CNO at 1...
Relevance to the DREADD system: is CNO an inert ligand?
reagent used in the protocol.
- Use
- The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itself or to the conversion to clozapine and N -Des, the administration of CNO is not without consequence. However, perhaps the biggest concern...
Solid-phase extraction
reagent used in the protocol.
- Use
- C2 extraction columns (100 mg/ml; ISOLUTE column, Biotage) were conditioned by sequential washing with 0.5 ml of elution solution (10 m m acetic acid, 5 m m trimethylamine), 3 × 1 ml methanol and 2 × 1 ml buffer solution rinses (100 m m sodium phosphate dihydrate, pH 4.6). Plasma (900 µl) was loaded t...
Relevance to the DREADD system: is CNO an inert ligand?
reagent used in the protocol.
- Use
- Despite the issues raised, the present findings do not render CNO unusable as an activating ligand in the DREADD system. Rather, it highlights the necessity for incorporation of the appropriate controls and careful consideration of the doses to be administered. An experimental design that includes a group of animals...
Relevance to the DREADD system: is CNO an inert ligand?
reagent used in the protocol.
- Use
- Our experiments were conducted in adult male Long-Evans rats, which, in addition to being a common outbred strain used in many behavioral experiments, are also the genetic background of the pTH:cre and pChAT:cre rats developed by. The generalization of our metabolism results to other rat strains, or to mice,...
Significance Statement
Recently, interest in clozapine N-oxide (CNO) has increased due to its exploitation as a ligand for the engineered G-protein-coupled receptors (GPCRs) in the chemogenetic 'Designer Receptors Exclusively Activated by Designer Drugs' (DREADD) system. Our results highlight that in the experimental design th...
- Use
- Recently, interest in clozapine N-oxide (CNO) has increased due to its exploitation as a ligand for the engineered G-protein-coupled receptors (GPCRs) in the chemogenetic 'Designer Receptors Exclusively Activated by Designer Drugs' (DREADD) system. Our results highlight that in the experimental design th...
Behavioral testing
Testing was conducted in startle chambers (Kinder-Scientific). Each sound-attenuating test chamber was equipped with small chambers mounted on a parallelogram load cell (calibrated to newtons) situated directly beneath a loudspeaker. Constant dim illumination was provided by a light within the chamber. Throughout te...
- Use
- Testing was conducted in startle chambers (Kinder-Scientific). Each sound-attenuating test chamber was equipped with small chambers mounted on a parallelogram load cell (calibrated to newtons) situated directly beneath a loudspeaker. Constant dim illumination was provided by a light within the chamber. Throughout te...
Locomotor testing
Locomotor testing was conducted in plastic cages measuring 45 × 23 × 20 cm interfaced by a grid array of infrared beams connected to a computer system that tracked and quantified the location and movements of the animal (OMNITECH Instruments). Rats were habituated to the testing room in their home cages fo...
- Use
- Locomotor testing was conducted in plastic cages measuring 45 × 23 × 20 cm interfaced by a grid array of infrared beams connected to a computer system that tracked and quantified the location and movements of the animal (OMNITECH Instruments). Rats were habituated to the testing room in their home cages fo...
Fast-Scan Cyclic Voltammetry
Rats were anesthetized with urethane (1.5-2.0 g/kg), placed into a stereotaxic apparatus, and implanted with a carbon fiber microelectrode aimed at the NAcc [+1.3 anterior, +1.3 lateral (L), -6.5 ventral (V), relative to bregma] and an Ag/AgCl reference electrode located in the contralateral cortex ( )....
- Use
- Rats were anesthetized with urethane (1.5-2.0 g/kg), placed into a stereotaxic apparatus, and implanted with a carbon fiber microelectrode aimed at the NAcc [+1.3 anterior, +1.3 lateral (L), -6.5 ventral (V), relative to bregma] and an Ag/AgCl reference electrode located in the contralateral cortex ( )....
Data acquisition
The electrode potential was linearly scanned (0.4-1.2 V and back to -0.4 V vs Ag/AgCl), and cyclic voltammograms were recorded at the carbon fiber electrode every 100 ms with a scan rate of 400 V/s using a voltammeter/amperometer (Chem-Clamp, Dagan Corporation). The magnitude of stimulated DA release and...
- Use
- The electrode potential was linearly scanned (0.4-1.2 V and back to -0.4 V vs Ag/AgCl), and cyclic voltammograms were recorded at the carbon fiber electrode every 100 ms with a scan rate of 400 V/s using a voltammeter/amperometer (Chem-Clamp, Dagan Corporation). The magnitude of stimulated DA release and...
HPLC studies
Clozapine (assay 98.9%) was obtained from MP Biomedicals. N -desmethylclozapine (assay >99%) and doxipine HCL internal standard (assay >99%) were obtained from Tocris Bioscience. For these studies, only CNO 5 mg/kg was tested because it was efficacious in the voltammetry and locomotor studies. In addition, CNO at 1...
- Use
- Clozapine (assay 98.9%) was obtained from MP Biomedicals. N -desmethylclozapine (assay >99%) and doxipine HCL internal standard (assay >99%) were obtained from Tocris Bioscience. For these studies, only CNO 5 mg/kg was tested because it was efficacious in the voltammetry and locomotor studies. In addition, CNO at 1...
Relevance to the DREADD system: is CNO an inert ligand?
The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itself or to the conversion to clozapine and N -Des, the administration of CNO is not without consequence. However, perhaps the biggest concern...
- Use
- The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itself or to the conversion to clozapine and N -Des, the administration of CNO is not without consequence. However, perhaps the biggest concern...
Relevance to the DREADD system: is CNO an inert ligand?
Despite the issues raised, the present findings do not render CNO unusable as an activating ligand in the DREADD system. Rather, it highlights the necessity for incorporation of the appropriate controls and careful consideration of the doses to be administered. An experimental design that includes a group of animals...
- Use
- Despite the issues raised, the present findings do not render CNO unusable as an activating ligand in the DREADD system. Rather, it highlights the necessity for incorporation of the appropriate controls and careful consideration of the doses to be administered. An experimental design that includes a group of animals...
Data analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Behavioral and neurochemical data were analyzed in SPSS version 22 (IBM). Details of individual tests are described within the relevant section in Results. Where graphs are displayed, these depict group means ± SEM. Results were considered statistically significant when p < 0.05. On graphs, * indicates signific...
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Significance Statement
Recently, interest in clozapine N-oxide (CNO) has increased due to its exploitation as a ligand for the engineered G-protein-coupled receptors (GPCRs) in the chemogenetic 'Designer Receptors Exclusively Activated by Designer Drugs' (DREADD) system. Our results highlight that in the experimental design there is a necessity for the inclusion of a group of animals which do not express DREADDs, but are given the same dose of CNO as the DREADD expressing animals. Currently, only a small minority of studies using DREADDs employ this control. There needs to be careful consideration of the CNO dose being administered and of the possible biological effects of CNO.
Materials and Methods
Male Long-Evans rats (Harlan Laboratories; rats were bred in our facilities) weighing 300-330 g at the start of behavioral studies and 300-450 g for voltammetric studies were maintained single housed in plastic cages in a temperature- and humidity-controlled room. Lights were on a 12 h light/dark cycle (lights on at 7:00 A.M.) with testing conducted during the light phase. Rats had free access to food (Harlan Diet 2018, Harlan Laboratories) and water in the home cage. All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Materials and Methods
CNO was supplied by the National Institutes of Health Drug Supply Program, and was dissolved in DMSO then diluted to a final concentration of 1, 2, or 5 mg/ml CNO in 0.5% DMSO in saline solution. Control injections were 0.5% DMSO in saline solution. Of the 22 articles that we found that used systemic administration of CNO in rats (;;;;;;;;;;;;;;;;;;;; ), 2 used chronic treatment (e.g., in drinking water) and 11 used doses >1 mg/kg. Of those that used doses of >1 mg/kg, most used doses of 3 mg/kg, but in a few cases doses were as high as 10 mg/kg. Of all the studies using rats and CNO-activated DREADDs that we surveyed, only a few used a non-DREADD CNO control, and most of these used this control in only a subset of the presented experiments. Our selection of 1, 2, and 5 mg/kg doses was based on our preliminary experience with CNO and the few published rat studies t...
Materials and Methods
Phencyclidine (PCP; Sigma-Aldrich), d -amphetamine (Sigma-Aldrich), and scopolamine (Tocris Bioscience) were dissolved in 0.9% saline solution, and injected at 1 ml/kg.
Behavioral testing
Following 5 min of acclimatization and the presentation of three 120 dB 40 ms pulses with a mean 15 s ITI, rats were exposed to eight trial types, presented 10 times each in a pseudorandom order with a mean ITI of 15 s (range, 5-25 s). Trial types were as follows: startle stimulus only (120, 110, 100, and 90 dB; 40 ms white noise) and four different prepulse plus pulse trials (68, 72, 76, and 80 dB prepulse, 20 ms duration, the onset of which was followed 120 ms later by a 120 db 40 ms pulse).
CNO-alone studies
To assess the effects of CNO on startle and PPI, rats were injected with CNO (1 mg/kg, i.p.) or 0.5% DMSO in saline solution 20 min before being placed in the PPI chambers. This dose was chosen based on the literature (; ) and consultation with investigators who had experience using DREADDs.
Phencyclidine studies
Knowing the possibility of CNO retroconversion, it was decided to test higher doses of CNO in a bioassay known to be sensitive to the presence of several antipsychotic agents, the disruption of prepulse inhibition by the NMDA antagonist PCP (; ). To assess the effects of CNO on PCP-induced disruption of PPI, rats were assigned to one of the following six experimental groups, each group containing two treatments: (1) vehicle (Veh) and vehicle; (2) vehicle and PCP; (3) CNO 2 mg/kg and vehicle; (4) CNO 5 mg/kg and vehicle; (5) CNO 2 mg/kg and PCP; and (6) CNO 5 mg/kg and PCP. Rats were injected with the first treatment (0.5% saline, i.p., in DMSO or CNO) followed 20 min later by the second treatment (saline or PCP 2.5 mg/kg, both s.c.). They were placed in the PPI chambers 10 min after the PCP injection. Due to there being no significant reversal of PCP-mediated disruption at CNO doses...
Scopolamine studies
To assess the effects of CNO on scopolamine-induced disruption of PPI, rats were assigned to one of the following four groups, each containing two treatments: (1) vehicle and vehicle; (2) vehicle and scopolamine; (3) CNO 5 mg/kg and vehicle; and (4) CNO 5 mg/kg and scopolamine. Rats were injected with the first treatment (0.5% DMSO in saline or 5 mg/kg CNO, both i.p.) followed 20 min later by the second treatment (saline or 0.5 mg/kg scopolamine, both s.c.). They were placed in the PPI chambers 10 min after the scopolamine injection. In light of the results with PCP, initial studies used 5 mg/kg CNO, and, because no significant reversal was observed, further doses were not evaluated.
Measurement outputs
What raw and processed outputs should exist?
Testing was conducted in startle chambers (Kinder-Scientific). Each sound-attenuating test chamber was equipped with small chambers mounted on a parallelogram load cell (calibra...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The electrode potential was linearly scanned (0.4-1.2 V and back to -0.4 V vs Ag/AgCl), and cyclic voltammograms were recorded at the carbon fiber electrode every 10...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itse...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
HPLC analysis was performed by a modification of the method described by on a Shimadzu Scientific Instruments Prominence 20A series HPLC and SPDM20A photodiode array. Stock solu...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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
Knowing the possibility of CNO retroconversion, it was decided to test higher doses of CNO in a bioassay known to be sensitive to the presence of several antipsychotic agents, the disruption of prepulse inhibition by the NMDA antagonist PCP (; ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Testing was conducted in startle chambers (Kinder-Scientific). Each sound-attenuating test chamber was equipped with small chambers mounted on a parallelogram load cell (calibra...; The electrode potential was linearly scanned (0.4-1.2 V and back to -0.4 V vs Ag/AgCl), and cyclic voltammograms were recorded at the carbon fiber electrode every 10...; The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itse...; HPLC analysis was performed by a modification of the method described by on a Shimadzu Scientific Instruments Prominence 20A series HPLC and SPDM20A photodiode array. Stock solu....
from paperStatistical comparison
Knowing the possibility of CNO retroconversion, it was decided to test higher doses of CNO in a bioassay known to be sensitive to the presence of several antipsychotic agents, t...; To assess the effects of CNO on scopolamine-induced disruption of PPI, rats were assigned to one of the following four groups, each containing two treatments: (1) vehicle and ve...; The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itse...; Behavioral and neurochemical data were analyzed in SPSS version 22 (IBM). Details of individual tests are described within the relevant section in Results. Where graphs are disp...
from paperReporting output
Report representative outputs alongside summary comparisons for Testing was conducted in startle chambers (Kinder-Scientific). Each sound-attenuating test chamber was equipped with small chambers mounted on a parallelogram load cell (calibra..., The electrode potential was linearly scanned (0.4-1.2 V and back to -0.4 V vs Ag/AgCl), and cyclic voltammograms were recorded at the carbon fiber electrode every 10..., The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itse..., HPLC analysis was performed by a modification of the method described by on a Shimadzu Scientific Instruments Prominence 20A series HPLC and SPDM20A photodiode array. Stock solu....
inferred from protocolStructured statistical methods
Knowing the possibility of CNO retroconversion, it was decided to test higher doses of CNO in a bioassay known to be sensitive to the presence of several antipsychotic agents, t...; To assess the effects of CNO on scopolamine-induced disruption of PPI, rats were assigned to one of the following four groups, each containing two treatments: (1) vehicle and ve...; The results from these studies show that CNO, in male Long-Evans rats, is not an inert ligand. Regardless of whether the behavioral effects we observed are due to CNO itse...; Behavioral and neurochemical data were analyzed in SPSS version 22 (IBM). Details of individual tests are described within the relevant section in Results. Where graphs are disp...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Recently, interest in clozapine N-oxide (CNO) has increased due to its exploitation as a ligand for the engineered G-protein-coupled receptors (GPCRs) in the chemogenetic 'Designer Receptors Exclusively Activated by Designer Drugs' (DREADD) system. Our results highlight that in the experimental design there is a necessity for the inclusion of a group of animals which do not express DREADDs, but are given the same dose of CNO as the DREADD expressing animals. Currently, only a small minority of studies using DREADDs employ this control. There needs to be careful consideration of the CNO dose being administered and of the possible biological effects of CNO.
Male Long-Evans rats (Harlan Laboratories; rats were bred in our facilities) weighing 300-330 g at the start of behavioral studies and 300-450 g for voltammetric studies were maintained single housed in plastic cages in a temperature- and humidity-controlled room. Lights were on a 12 h light/dark cycle (lights on at 7:00 A.M.) with testing conducted during the light phase. Rats had free access to food (Harlan Diet 2018, Harlan Laboratories) and water in the home cage. All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
CNO was supplied by the National Institutes of Health Drug Supply Program, and was dissolved in DMSO then diluted to a final concentration of 1, 2, or 5 mg/ml CNO in 0.5% DMSO in saline solution. Control injections were 0.5% DMSO in saline solution. Of the 22 articles that we found that used systemic administration of CNO in rats (;;;;;;;;;;;;;;;;;;;; ), 2 used chronic treatment (e.g., in drinking water) and 11 used doses >1 mg/kg. Of those that used doses of >1 mg/kg, most used doses of 3 mg/kg, but in a few cases doses were as high as 10 mg/kg. Of all the studies using rats and CNO-activated DREADDs that we surveyed, only a few used a non-DREADD CNO control, and most of these used this control in only a subset of the presented experiments. Our selection of 1, 2, and 5 mg/kg doses was based on our preliminary experience with CNO and the few published rat studies that were available at the time. Also, Dr. Roth's research group had shown that in mice there were no effects at doses of 5 mg/kg ( ) and that some mouse studies had used doses as high as 10 mg/kg ( ).
Phencyclidine (PCP; Sigma-Aldrich), d -amphetamine (Sigma-Aldrich), and scopolamine (Tocris Bioscience) were dissolved in 0.9% saline solution, and injected at 1 ml/kg.
Following 5 min of acclimatization and the presentation of three 120 dB 40 ms pulses with a mean 15 s ITI, rats were exposed to eight trial types, presented 10 times each in a pseudorandom order with a mean ITI of 15 s (range, 5-25 s). Trial types were as follows: startle stimulus only (120, 110, 100, and 90 dB; 40 ms white noise) and four different prepulse plus pulse trials (68, 72, 76, and 80 dB prepulse, 20 ms duration, the onset of which was followed 120 ms later by a 120 db 40 ms pulse).
To assess the effects of CNO on startle and PPI, rats were injected with CNO (1 mg/kg, i.p.) or 0.5% DMSO in saline solution 20 min before being placed in the PPI chambers. This dose was chosen based on the literature (; ) and consultation with investigators who had experience using DREADDs.
Knowing the possibility of CNO retroconversion, it was decided to test higher doses of CNO in a bioassay known to be sensitive to the presence of several antipsychotic agents, the disruption of prepulse inhibition by the NMDA antagonist PCP (; ). To assess the effects of CNO on PCP-induced disruption of PPI, rats were assigned to one of the following six experimental groups, each group containing two treatments: (1) vehicle (Veh) and vehicle; (2) vehicle and PCP; (3) CNO 2 mg/kg and vehicle; (4) CNO 5 mg/kg and vehicle; (5) CNO 2 mg/kg and PCP; and (6) CNO 5 mg/kg and PCP. Rats were injected with the first treatment (0.5% saline, i.p., in DMSO or CNO) followed 20 min later by the second treatment (saline or PCP 2.5 mg/kg, both s.c.). They were placed in the PPI chambers 10 min after the PCP injection. Due to there being no significant reversal of PCP-mediated disruption at CNO doses of 2 or 5 mg/kg, lower doses were not pursued.
To assess the effects of CNO on scopolamine-induced disruption of PPI, rats were assigned to one of the following four groups, each containing two treatments: (1) vehicle and vehicle; (2) vehicle and scopolamine; (3) CNO 5 mg/kg and vehicle; and (4) CNO 5 mg/kg and scopolamine. Rats were injected with the first treatment (0.5% DMSO in saline or 5 mg/kg CNO, both i.p.) followed 20 min later by the second treatment (saline or 0.5 mg/kg scopolamine, both s.c.). They were placed in the PPI chambers 10 min after the scopolamine injection. In light of the results with PCP, initial studies used 5 mg/kg CNO, and, because no significant reversal was observed, further doses were not evaluated.
Machine-readable layer
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"name": "Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments methods",
"description": "Evidence-backed execution summary for Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments methods from Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments.",
"totalTime": "PT65M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Significance Statement",
"text": "Recently, interest in clozapine N-oxide (CNO) has increased due to its exploitation as a ligand for the engineered G-protein-coupled receptors (GPCRs) in the chemogenetic 'Designer Receptors Exclusively Activated by Designer Drugs' (DREADD) system. Our results highlight that in the experimental design there is a necessity for the inclusion of a group of animals which do not express DREADDs, but are given the same dose of CNO as the DREADD expressing animals. Currently, only a small minority of studies using DREADDs employ this control. There needs to be careful consideration of the CNO dose being administered and of the possible biological effects of CNO."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Materials and Methods",
"text": "Male Long-Evans rats (Harlan Laboratories; rats were bred in our facilities) weighing 300-330 g at the start of behavioral studies and 300-450 g for voltammetric studies were maintained single housed in plastic cages in a temperature- and humidity-controlled room. Lights were on a 12 h light/dark cycle (lights on at 7:00 A.M.) with testing conducted during the light phase. Rats had free access to food (Harlan Diet 2018, Harlan Laboratories) and water in the home cage. All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Materials and Methods",
"text": "CNO was supplied by the National Institutes of Health Drug Supply Program, and was dissolved in DMSO then diluted to a final concentration of 1, 2, or 5 mg/ml CNO in 0.5% DMSO in saline solution. Control injections were 0.5% DMSO in saline solution. Of the 22 articles that we found that used systemic administration of CNO in rats (;;;;;;;;;;;;;;;;;;;; ), 2 used chronic treatment (e.g., in drinking water) and 11 used doses >1 mg/kg. Of those that used doses of >1 mg/kg, most used doses of 3 mg/kg, but in a few cases doses were as high as 10 mg/kg. Of all the studies using rats and CNO-activated DREADDs that we surveyed, only a few used a non-DREADD CNO control, and most of these used this control in only a subset of the presented experiments. Our selection of 1, 2, and 5 mg/kg doses was based on our preliminary experience with CNO and the few published rat studies t..."
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},
{
"@type": "HowToStep",
"position": 5,
"name": "Behavioral testing",
"text": "Following 5 min of acclimatization and the presentation of three 120 dB 40 ms pulses with a mean 15 s ITI, rats were exposed to eight trial types, presented 10 times each in a pseudorandom order with a mean ITI of 15 s (range, 5-25 s). Trial types were as follows: startle stimulus only (120, 110, 100, and 90 dB; 40 ms white noise) and four different prepulse plus pulse trials (68, 72, 76, and 80 dB prepulse, 20 ms duration, the onset of which was followed 120 ms later by a 120 db 40 ms pulse)."
},
{
"@type": "HowToStep",
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"name": "CNO-alone studies",
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},
{
"@type": "HowToStep",
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"name": "Phencyclidine studies",
"text": "Knowing the possibility of CNO retroconversion, it was decided to test higher doses of CNO in a bioassay known to be sensitive to the presence of several antipsychotic agents, the disruption of prepulse inhibition by the NMDA antagonist PCP (; ). To assess the effects of CNO on PCP-induced disruption of PPI, rats were assigned to one of the following six experimental groups, each group containing two treatments: (1) vehicle (Veh) and vehicle; (2) vehicle and PCP; (3) CNO 2 mg/kg and vehicle; (4) CNO 5 mg/kg and vehicle; (5) CNO 2 mg/kg and PCP; and (6) CNO 5 mg/kg and PCP. Rats were injected with the first treatment (0.5% saline, i.p., in DMSO or CNO) followed 20 min later by the second treatment (saline or PCP 2.5 mg/kg, both s.c.). They were placed in the PPI chambers 10 min after the PCP injection. Due to there being no significant reversal of PCP-mediated disruption at CNO doses..."
},
{
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"name": "Scopolamine studies",
"text": "To assess the effects of CNO on scopolamine-induced disruption of PPI, rats were assigned to one of the following four groups, each containing two treatments: (1) vehicle and vehicle; (2) vehicle and scopolamine; (3) CNO 5 mg/kg and vehicle; and (4) CNO 5 mg/kg and scopolamine. Rats were injected with the first treatment (0.5% DMSO in saline or 5 mg/kg CNO, both i.p.) followed 20 min later by the second treatment (saline or 0.5 mg/kg scopolamine, both s.c.). They were placed in the PPI chambers 10 min after the scopolamine injection. In light of the results with PCP, initial studies used 5 mg/kg CNO, and, because no significant reversal was observed, further doses were not evaluated."
}
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