Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments methods
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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.
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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.
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