Clozapine n oxide dihydrochloride

Manufactured by Bio-Techne

Clozapine N-oxide dihydrochloride is a synthetic compound. It is a salt of clozapine N-oxide, which is an oxidized metabolite of the antipsychotic drug clozapine.

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10 protocols using clozapine n oxide dihydrochloride

Pharmacological Modulation of Neurotransmission

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Most drugs were dissolved in water to make stock solutions (typically 10,000x concentrations) that were frozen as aliquots and used as needed during experiments. Acetylcholine (Fisher Scientific) was bath-applied with physostigmine hemisulfate (eserine; Tocris Bioscience), a blocker of Acetylcholinesterase. Atropine and pirenzepine dihydrochloride were purchased from Sigma-Aldrich. Tetrodotoxin (TTX) citrate and CGP 52432 were purchased from HelloBio, Inc. Clozapine N-oxide (CNO) dihydrochloride was purchased from Tocris Biosciences. SR-95531 (gabazine) was purchased from MedChemExpress and dissolved in DMSO for stock solutions.

, & Gulledge A.T. (2023). Cholinergic activation of corticofugal circuits in the adult mouse prefrontal cortex. bioRxiv.

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Activating Excitatory DREADD hM3Dq

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Clozapine N‐oxide (CNO) dihydrochloride (Tocris) was dissolved in physiological saline to 0.03 mg/ml. For activation of the excitatory DREADD hM3Dq, the CNO solution was injected through intraperitoneal administration route at a concentration of 0.3 mg/10 ml/kg at 30 min prior to the beginning of each behavioral session or transcardial perfusion.

Kim B, & Im H. (2020). Chronic nicotine impairs sparse motor learning via striatal fast‐spiking parvalbumin interneurons. Addiction Biology, 26(3), e12956.

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Pharmacological Interventions in Rodent Models

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Cocaine HCl (from the National Institute on Drug Abuse) was diluted in 0.9% NaCl saline solution (ICU Medical) and injected intraperitoneally at 20, 10 or 5 mg/kg. Morphine SO₄ (from the National Institute on Drug Abuse) was diluted in 0.9% NaCl saline solution (ICU Medical) and injected subcutaneously at 7.5 mg/kg. HX531 (Tocris, Cat. No. 3912) was first diluted in DMSO (Sigma), then in Dulbecco’s Phosphate Buffered Saline (PBS, Gibco) to a 1% DMSO final concentration and injected intraperitoneally 20 min before testing at 20 mg/kg in mice, and to a 5% DMSO final concentration and injected intraperitoneally 30 min before testing at 12 mg/kg in rats. For chemogenetics, clozapine-N-oxide (CNO) dihydrochloride (Tocris, #6329) was diluted in PBS and injected intraperitoneally 15 min before testing at 2 mg/kg.

Godino A., Salery M., Durand-de Cuttoli R., Estill M.S., Holt L.M., Futamura R., Browne C.J., Mews P., Hamilton P.J., Neve R.L., Shen L., Russo S.J, & Nestler E.J. (2023). Transcriptional control of nucleus accumbens neuronal excitability by Retinoid X Receptor Alpha tunes sensitivity to drug rewards. Neuron, 111(9), 1453-1467.e7.

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Pharmacological Manipulation of Stress

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Imipramine (I0899, Sigma-Aldrich), corticosterone (C2505, Sigma-Aldrich), NBI27914 hydrochloride (1591, Tocris Bioscience), RU486 (M8046, Sigma-Aldrich), and Clozapine N-oxide (CNO) dihydrochloride (6329, Tocris Bioscience) were diluted in 0.9 % saline and injected intraperitoneally in a volume of 120 μl.

Lee E.H., Park J.Y., Kwon H.J, & Han P.L. (2021). Repeated exposure with short-term behavioral stress resolves pre-existing stress-induced depressive-like behavior in mice. Nature Communications, 12, 6682.

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Chemogenetic Manipulation of Mouse Behavior

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Viral pAAV-hsyn-DIO-hm4D(Gi) (Addgene_44362-AAV5) injected mice were administered intraperitoneally with Clozapine N-oxide dihydrochloride (CNO, 2mg/kg, Tocris) ten minutes before the licking or tail shocking task. Only the mice reached stable behavioral level (after at least two test phases and (LO – DO < 3s) in all test phases) were used for chemogenetic experiments. In the licking task, mice were re-trained to lick the water one to two times after recovery from CNO administration. The re-trained phases were not included in test phases.

Wang Y, & Sun Q.Q. (2023). Persistence is driven by a prefrontal motor circuit. Research Square.

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Modulating Neuronal Activity in GAD2-Cre Mice

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On recording days, saline or CNO (5 mg/kg, clozapine N-oxide dihydrochloride, Tocris Bioscience) was injected intraperitoneally (i.p.) into GAD2-Cre mice expressing hM4D(Gi) or mCherry in the dmM. Injections occurred at 10:30 am. Each recording session began 30 min after injection and each animal contributed 2 saline and 2 CNO recording sessions to the data set. The order of injection days (saline vs CNO) was randomly assigned. For statistical analysis, we compared the impact of CNO or saline in hM4D(Gi) or mCherry-expressing mice on the brain state using mixed ANOVA with virus (mCherry vs hM4D(Gi)) and drug (saline vs CNO) as within and between factors, followed by pairwise t-tests with Bonferroni correction.

Stucynski J.A., Schott A.L., Baik J., Chung S, & Weber F. (2021). Regulation of REM Sleep by Inhibitory Neurons in the Dorsomedial Medulla. Current biology : CB, 32(1), 37-50.e6.

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Chemogenetic Experiments with EEG/EMG/LFP

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For chemogenetic experiments, animals were tethered to a flexible cable for EEG/EMG/LFP recordings^{20 (link)}. On recording days, mice received intraperitoneal (i.p.) injections of saline or CNO (Clozapine N-oxide dihydrochloride, Tocris, Cat. No. 6329). Mice expressing hM3D(Gq) in the dmM received 0.25 mg/kg CNO, while mice expressing hM4D(Gi) received a dose of 5 mg/kg. Recording sessions began immediately after injection, and each animal contributed 2 saline and 2 CNO recordings to the dataset. mCherry control mice received both CNO doses, and underwent 4 saline and 4 CNO recording sessions in total. The order of injection days (saline vs CNO) was randomly determined.

Schott A.L., Baik J., Chung S, & Weber F. (2023). A medullary hub for controlling REM sleep and pontine waves. Nature Communications, 14, 3922.

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Chemogenetic Manipulation of Mice Behavior

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Viral pAAV-hsyn-DIO-hm4D(Gi) (Addgene_44362-AAV5) injected mice were administered intraperitoneally with Clozapine N-oxide dihydrochloride (CNO, 2mg kg⁻¹, Tocris) ten minutes before the licking or tail shocking task. Only the mice reached stable behavioral level (after at least two test phases and (LO – DO < 3s) in all test phases) were used for chemogenetic experiments. In the licking task, mice were re-trained to lick the water one to two times after recovery from CNO administration. The re-trained phases were not included in test phases.

Wang Y, & Sun Q.Q. (2024). A prefrontal motor circuit initiates persistent movement. bioRxiv.

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Neuropharmacology Protocol: In Vitro and In Vivo Assays

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Urethane (U2500, Merck), Fenfluramine ((+)-Fenfluramine hydrochloride, F112, Merck), 5-HT (Serotonin creatinine sulfate monohydrate, H7752, Merck), m-CPBG (1-(3-Chlorophenyl)biguanide hydrochloride, C144, Merck), tropisetron (Tropisetron hydrochloride,Y0000616, Sigma), WAY-100635 (W108, Merck), α-Methylserotonin (α-Methylserotonin maleate salt, M110, Merck), MDMA ((±)3,4-methylenedioxymethamphetamine, 64057-70-1, Merck), CNO (Clozapine N-oxide dihydrochloride, 6329, Tocris) were dissolved in water for in vitro application and in 0.9% normal saline for in vivo application. Ketanserin (Ketanserin (+)-tartrate salt, S006, Merck) was dissolved in Dimethyl sulfoxide (DMSO).

de Filippo R., Rost B.R., Stumpf A., Cooper C., Tukker J.J., Harms C., Beed P, & Schmitz D. (2021). Somatostatin interneurons activated by 5-HT2A receptor suppress slow oscillations in medial entorhinal cortex. eLife, 10, e66960.

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Chemogenetic Inhibition of CA1 during Training

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To chemogenetically inhibit CA1 during training, a Gi coupled DREADD (AAV9-hSyn- hM4D(Gi)-mCherry) virus was injected in CA1 (AAV9-hSyn-mCherry for controls) bilaterally along with injection of AAV9-hSyn-GCaMP6f into AC. Craniotomy and coverslip implant was performed on the mice two weeks after injection. Mice were water deprived and habituated after two weeks of recovery. 5mg/kg Clozapine N-oxide dihydrochloride (Tocris; Cat. No. 6329) was delivered intraperitoneally to both control (mCherry) and inhibited (hM4DGi) cohorts throughout training days 1–3. Retrieval was performed the following day. Mice were imaged on training day 3 and retrieval day 1.

Yadav N., Noble C., Niemeyer J.E., Terceros A., Victor J., Liston C, & Rajasethupathy P. (2022). Prefrontal Feature Representations Drive Memory Recall. Nature, 608(7921), 153-160.

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