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Clozapine n oxide dihydrochloride

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Clozapine N-oxide (CNO) dihydrochloride is a synthetic compound used as a research tool in neuroscience studies. It is a selective agonist for the designer receptor exclusively activated by designer drugs (DREADD) system, which allows for remote control of neuronal activity in experimental settings.

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Lab products found in correlation

Scopolamine hydrobromide, by Bio-Techne (2 mentions) Clozapine dihydrochloride, by Hello Bio (2 mentions) Oxotremorine m, by Bio-Techne (2 mentions) S5007, by Merck Group (1 mentions) Minocycline hcl, by Merck Group (1 mentions) Tamoxifen, by Merck Group (1 mentions) Deschloroclozapine dihydrochloride dcz, by Hello Bio (1 mentions) Naltrexone hydrochloride, by Merck Group (1 mentions) Naloxone hydrochloride, by Bio-Techne (1 mentions)

11 protocols using clozapine n oxide dihydrochloride

1

Chemogenetic Manipulation of Cocaine Reward

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During surgery, AAV5-hSyn-DIO-hM4D(Gi)-mCherry (Princeton Viral Core, injected 1 uL per hemisphere at a titer of 3.0 × 1013 genome copies/mL) was infused bilaterally in ventral medial NAc (M-L, +/− 0.65 mm; A-P, 1.43 mm; D-V, 4.75 mm) of experimental mice. Control mice received bilateral infusions of AAV2-EF1a-DIO-mCherry (Princeton Viral Core, injected 1 uL per hemisphere at a titer of 3.8 × 1013 genome copies/mL) at the same coordinates in ventral medial NAc. Animals were anesthetized for surgeries with isofluorane (3–4% induction and 1–2% maintenance). Behavioral testing occurred 3–4 weeks after surgery to allow for animal recovery and viral expression. During a repeat extinction cocaine CPP behavior experiment (see Cocaine conditioned place preference), both experimental and control mice received injections of clozapine N-oxide (CNO dihydrochloride; 3 mg/kg, i.p.; HelloBio) 30 min before each Test session.
Fleming W., Lee J., Briones B.A., Bolkan S.S, & Witten I.B. (2022). Cholinergic interneurons mediate cocaine extinction in male mice through plasticity across medium spiny neuron subtypes. Cell reports, 39(9), 110874.
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2

Chemogenetic Manipulation of Cocaine Reward

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During surgery, AAV5-hSyn-DIO-hM4D(Gi)-mCherry (Princeton Viral Core, injected 1 uL per hemisphere at a titer of 3.0 × 1013 genome copies/mL) was infused bilaterally in ventral medial NAc (M-L, +/− 0.65 mm; A-P, 1.43 mm; D-V, 4.75 mm) of experimental mice. Control mice received bilateral infusions of AAV2-EF1a-DIO-mCherry (Princeton Viral Core, injected 1 uL per hemisphere at a titer of 3.8 × 1013 genome copies/mL) at the same coordinates in ventral medial NAc. Animals were anesthetized for surgeries with isofluorane (3–4% induction and 1–2% maintenance). Behavioral testing occurred 3–4 weeks after surgery to allow for animal recovery and viral expression. During a repeat extinction cocaine CPP behavior experiment (see Cocaine conditioned place preference), both experimental and control mice received injections of clozapine N-oxide (CNO dihydrochloride; 3 mg/kg, i.p.; HelloBio) 30 min before each Test session.
Fleming W., Lee J., Briones B.A., Bolkan S.S, & Witten I.B. (2022). Cholinergic interneurons mediate cocaine extinction in male mice through plasticity across medium spiny neuron subtypes. Cell reports, 39(9), 110874.
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3

Preparation and Administration of 4-OHT and CNO

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Stock solution of 4-hydroxytamoxifen (4-OHT; H6278, Sigma Aldrich) was prepared by diluting 50 mg of 4-OHT in 2 ml of 100% ethanol. The solution was warmed on a heating platform at 37°C and vortexed until precipitates dissolved. The solution was then aliquoted and stored at −20°C until use (maximum 8 weeks in advance). On the day of the experiment, aliquots were defrosted on a heating platform at 37°C and vortexed until precipitates dissolved. Sterilized corn oil was added to reach a concentration of 10 mg ml−1. The solution was then warmed on a heating platform at 37°C and repeatedly vortexed every 15 min for ~1-2 hrs until the ethanol was completely evaporated. 4-OHT was administered IP following habituation to experimental handling and IP saline injection in the days prior to testing.
Clozapine N-oxide (CNO) dihydrochloride (1 mg kg−1, Hello Bio, Inc.) was prepared in sterile 0.9% saline and stored in a dark compartment at room temperature until use (maximum 4 weeks in advance). CNO was administered IP following habituation to experimental handling and IP saline injection in the days prior to testing.
Crowell D.N., Kadlecek A.T., John M.C, & Amasino R.M. (1990). Cytokinin-induced mRNAs in cultured soybean cells.. Proceedings of the National Academy of Sciences of the United States of America, 87(22), 8815-8819.
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4

Intraperitoneal administration of CNO

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Clozapine N-oxide (CNO) dihydrochloride (Hello Bio) was dissolved in 0.9% saline and injected intraperitoneally 30 min before the start of the dark phase. Solutions were freshly prepared on each test day and doses were administered using a Latin square design. Animals received either 0, 5, or 10 mg/kg CNO per test day, based on previously reported CNO doses (Hart et al., 2018 (link); Panoz-Brown et al., 2018 (link)).
Bruinsma B., Pattij T, & Mansvelder H.D. (2022). Prefrontal Cortical to Mediodorsal Thalamus Projection Neurons Regulate Posterror Adaptive Control of Behavior. eNeuro, 9(6), ENEURO.0254-22.2022.
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5

Pharmacological Agents for Neuromodulation

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Clozapine N-oxide (CNO) dihydrochloride (water soluble) and clozapine dihydrochloride (water soluble) were purchased from Hello Bio Inc. (Princeton, NJ, USA). Scopolamine hydrobromide, Oxotremorine M, and aCSF were purchased from Tocris (Minneapolis, MN, USA). Stock solutions of drugs were made and working concentrations of the drugs were prepared freshly on the day of each experiment by appropriate dilutions of the stock solutions in aCSF.
Yavas E., Trott J.M, & Fanselow M.S. (2021). Sexually dimorphic muscarinic acetylcholine receptor modulation of contextual fear learning in the dentate gyrus. Neurobiology of learning and memory, 185, 107528.
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6

Pharmacological Interventions in Mice

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Ethanol solutions were prepared in tap water from 95 % ethyl alcohol (Decon Labs, catalog # 2801TP) and sucrose (Sigma Aldrich, catalog # BP220–212). Minocycline HCl (Sigma Aldrich, catalog # M9511) was dissolved in distilled water (0.0416 mg/mL) and administered via drinking bottles at 3 h into the dark cycle for an average daily dose of 7.1 ± 0.09 mg/kg/day. Tamoxifen (TM; Sigma Aldrich, catalog # T5648) was dissolved in Sunflowerseed Oil (Sigma Aldrich, catalog # S5007) and administered at a dose of 75 mg/kg (i.p.). Clozapine N-oxide (CNO) dihydrochloride (Hello Bio, catalog # HB6149) was dissolved in sterile saline and administered by intraperitoneal injection (3 mg/kg, i. p.) 45 min before behavioral testing. For pain management of mice undergoing surgery, animals were treated with meloxicam (0.4 mg/mL at 10 mL/kg, s.c.; Norbrook Laboratories Limited, NDC: 55529–040–10) immediately following, and 24 h after surgery.
Nahata M.C., Vashi V.I., Swanson R.N., Messig M.A, & Chung M. (1999). Pharmacokinetics of Ampicillin and Sulbactam in Pediatric Patients. Antimicrobial Agents and Chemotherapy, 43(5), 1225-1229.
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7

Pharmacological Agents for Neuromodulation

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Clozapine N-oxide (CNO) dihydrochloride (water soluble) and clozapine dihydrochloride (water soluble) were purchased from Hello Bio Inc. (Princeton, NJ, USA). Scopolamine hydrobromide, Oxotremorine M, and aCSF were purchased from Tocris (Minneapolis, MN, USA). Stock solutions of drugs were made and working concentrations of the drugs were prepared freshly on the day of each experiment by appropriate dilutions of the stock solutions in aCSF.
Yavas E., Trott J.M, & Fanselow M.S. (2021). Sexually dimorphic muscarinic acetylcholine receptor modulation of contextual fear learning in the dentate gyrus. Neurobiology of learning and memory, 185, 107528.
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8

Clozapine N-oxide Dosing in Rodents

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Two weeks before testing, animals were habituated to injections by giving them several saline injections. Directly before a testing session, Clozapine N-oxide (CNO) dihydrochloride (Hello Bio, UK) was dissolved in 0.9% saline and injected intraperitoneally (i.p.) 30 min prior to the start of the dark phase. Solutions were freshly prepared on each test day and doses were administered using a Latin square design. Animals received either 0, 5, or 10 mg/kg CNO per testing session, in randomized order, based on recent work in rats25 (link).
de Kloet S.F., Bruinsma B., Terra H., Heistek T.S., Passchier E.M., van den Berg A.R., Luchicchi A., Min R., Pattij T, & Mansvelder H.D. (2021). Bi-directional regulation of cognitive control by distinct prefrontal cortical output neurons to thalamus and striatum. Nature Communications, 12, 1994.
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9

Pharmacological Activation of Neuronal Circuits

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Clozapine-N-oxide dihydrochloride (CNO; Hello Bio, Princeton, NJ, USA) was dissolved in sterile, 0.9% saline to a concentration of 0.30 mg/ml and injected at 10 ml/kg, i.p., 30 min before the beginning of behavioral experiments for a dose of 3 mg/kg. For c-fos studies, CNO was injected 2 hours before perfusion and brain extraction. Deschloroclozapine dihydrochloride (DCZ; Hello Bio) was dissolved in sterile, 0.9% saline to a concentration of 10 μg/ml and injected at 10 ml/kg, i.p., 20 min before the beginning of behavioral experiments for a final dose of 100 μg/kg, which was previously found to induce neural activity in hM3Dq-expressing neurons in mice and monkeys17 (link).
Marcinkiewcz C., Wang R., Khan K., Balasubramanian N., James T., Pushpavathi S., Kim D., Pierson S., Wu Q., Niciu M, & Hefti M. (2023). Alcohol inhibits sociability via serotonin inputs to the nucleus accumbens. Research Square.
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10

DREADD-Mediated Modulation of Social Defeat

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Clozapine N-oxide dihydrochloride (CNO; 2 mg/kg; Cat# HB6149; HelloBio, Princeton, NJ) was diluted in in lactated ringers and delivered by intraperitoneal (IP) injections on each day of social defeat, where indicated (Figure 2A), between ZT1 and ZT2 (ZT12=lights off, early light phase). Mice expressing both control construct and excitatory DREADD received CNO. For validation studies (Figure 2C), single IP injections were delivered at ZT 10.
Bush B.J., Donnay C., Andrews E.J., Lewis-Sanders D., Gray C.L., Qiao Z., Brager A.J., Johnson H., Brewer H.C., Sood S., Saafir T., Benveniste M., Paul K.N, & Ehlen J.C. (2022). Non-rapid eye movement sleep determines resilience to social stress. eLife, 11, e80206.
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