Clozapine n oxide

Manufactured by Merck Group

Sourced in United States

Clozapine N-oxide is a chemical compound used as a laboratory tool. It is a structural analogue of the antipsychotic drug clozapine. Clozapine N-oxide is commonly used as a pharmacological tool to activate engineered G-protein coupled receptors in experimental settings.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by Merck Group (6 mentions) Aav hsyn dio mcherry, by Addgene (3 mentions) Aav hsyn dio hm3d gq mcherry, by Addgene (2 mentions) Isoflurane, by RWD Life Science (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Phenol red, by Thermo Fisher Scientific (2 mentions) Human recombinant insulin, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Merck Group (2 mentions) Cholera toxin, by Merck Group (2 mentions) Lncap, by American Type Culture Collection (2 mentions) Hydrocortisone, by Merck Group (2 mentions) Sodium selenite, by Merck Group (2 mentions) Thrombin, by Merck Group (2 mentions) Carbachol, by Merck Group (2 mentions) Mda pca 2b, by American Type Culture Collection (2 mentions) Lncap, by Merck Group (2 mentions) Vs 4718, by Selleck Chemicals (2 mentions) Mouse egf, by Corning (2 mentions) Enzalutamide, by Selleck Chemicals (2 mentions)

Spelling variants of this product

Clozapine (93 citations) Clozapine-N-oxide (CNO, (70 citations)

44 protocols using clozapine n oxide

Chemogenetic Activation of SST Neurons and Food Intake

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Clozapine-n-oxide (CNO; MilliporeSigma #0832) was used to activate TN^SST neurons in Sst-Cre animals expressing hM3Dq-mCherry. Stock solution of 20 mg/mL in DMSO was stored at -20°C and diluted to a working solution of 0.03 mg/mL in sterile saline also stored at -20°C. Saline control (0.15% DMSO) or CNO (10 μL/g body weight, dose of 0.3 mg/kg) working solution were administered IP in a counterbalanced design. Experiments were completed in duplicate replicate trials. Mice were transferred to experimental room at least 15 minutes prior to experimentation. Experiments were begun between ZT 2-3 and terminated between ZT 6-7. Following injection, food intake was measured at 0.5, 1, 2, and 4 hr. Vaginal lavage was performed on female mice after experiment conclusion to prevent stress interference with food intake. All mice were injected with CNO 90 minutes prior to sacrifice to enable neuronal activation validation via cFOS immunohistochemistry.

Massa M.G., Scott R.L., Cara A.L., Cortes L.R., Vander P.B., Sandoval N.P., Park J.W., Ali S.L., Velez L.M., Wang H.B., Ati S.S., Tesfaye B., Reue K., van Veen J.E., Seldin M.M, & Correa S.M. (2023). Feeding neurons integrate metabolic and reproductive states in mice. iScience, 26(10), 107918.

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Activating SST Neurons with Clozapine-n-oxide

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Clozapine-n-oxide (CNO; MilliporeSigma #0832) was used to activate TN^SST neurons in Sst-Cre animals expressing hM3Dq-mCherry. Stock solution of 20 mg/mL in DMSO was stored at −20°C and diluted to a working solution of 0.03 mg/mL in sterile saline also stored at −20°C. Saline control (0.15% DMSO) or CNO (10 μL/g body weight, dose of 0.3 mg/kg) working solution were administered IP in a counterbalanced design. Experiments were completed in duplicate replicate trials. Mice were transferred to experimental room at least 15 minutes prior to experimentation. Experiments were begun between ZT 2-3 and terminated between ZT 6-7. Following injection, food intake was measured at 0.5, 1, 2, and 4 hr. Vaginal lavage was performed on female mice after experiment conclusion to prevent stress interference with food intake. All mice were injected with CNO 90 minutes prior to sacrifice to enable neuronal activation validation via cFOS immunohistochemistry.

Massa M.G., Scott R.L., Cara A.L., Cortes L.R., Sandoval N.P., Park J.W., Ali S., Velez L.M., Tesfaye B., Reue K., van Veen J.E., Seldin M, & Correa S.M. (2023). Feeding Neurons Integrate Metabolic and Reproductive States in Mice. bioRxiv.

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Targeted Behavioral Measurements in Chemogenetic Manipulation

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Animals underwent behavioral training as above. To target behavioral measurements to a time period overlapping with circuit attenuation^{82 (link)} that also avoids indirect effects of agonist treatment,^{83 (link)} all mice were given intraperitoneal injections of the hM4Di selective agonist Clozapine-N-Oxide (CNO, 1.0 mg/kg, 10 ml/kg, Sigma Aldrich) 30 minutes prior to each hold down training session.

Schreiner D.C., Wright A., Baltz E.T., Wang T., Cazares C, & Gremel C.M. (2023). Chronic alcohol exposure alters action control via hyperactive premotor corticostriatal activity. Cell reports, 42(7), 112675.

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Cre-dependent Expression of Excitatory Designer Receptor

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For Cre-recombinase-dependent expression of the excitatory designer receptor hM3Dq in preoptic glutamatergic (Vglut2⁺) neurons, we used the adeno-associated viral vector AAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene; catalog #50459-AAV5) (Krashes et al., 2011 (link); Vanini et al., 2020 (link)). In a separate group of Vglut-Cre mice used for control experiments, we used the Cre-dependent vector AAV-hSyn-DIO-mCherry (Addgene; catalog #50459-AAV5) that lacked the coding sequence for the designer receptor and only contained the fluorescent reporter mCherry. The titer of the viral solutions was 3.7-7.8 × 10¹² copies per ml. DMSO and clozapine-N-oxide (CNO, catalog #C0832-5MG; agonist at hM3Dq receptors) were purchased from Sigma Millipore. A stock solution of CNO (0.1 mg/ml in saline solution containing 0.5% DMSO) was prepared and stored in aliquots, and then frozen at −20°C until use. Before each experiment, an aliquot of the stock solution was thawed at room temperature protected from the light until injection time. Mice received 1 mg/kg CNO or a vehicle control solution (VEH, saline with 0.5% DMSO) by intraperitoneal injection; the injection volume was 0.1 ml per 10 g of body weight (Vanini et al., 2020 (link)).

Mondino A., Hambrecht-Wiedbusch V.S., Li D., York A.K., Pal D., González J., Torterolo P., Mashour G.A, & Vanini G. (2021). Glutamatergic Neurons in the Preoptic Hypothalamus Promote Wakefulness, Destabilize NREM Sleep, Suppress REM Sleep, and Regulate Cortical Dynamics. The Journal of Neuroscience, 41(15), 3462-3478.

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Clozapine N-Oxide and OX2R Antagonist Effects

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Clozapine N-Oxide (CNO; Sigma–Aldrich; St Louis, MO, USA) was dissolved in saline and 8% DMSO and injected intraperitoneally at 2 mg/kg in a 5 mg/mL solution. This dose is in accordance with doses used in previous DREADDs studies in rats (Farrell and Roth, 2013 (link)). MK-1064, a selective OX₂R antagonist, (Roecker et al., 2014 (link)) was dissolved overnight in 20% Vitamin E d-a-tocopherol polyethylene glycol 1000 succinate (Vit E-TPGS) (Sigma–Aldrich; St Louis, MO, USA). Rats were orally administered either 20% Vit E-TPGS as vehicle or 30 mg/kg MK-1064, both at 1 mL/kg for consistent volume. All animals were given a vehicle dose 1 day prior to the start of the restraint paradigm to habituate to the oral gavage procedure. Animals received injections of vehicle (20% Vit E TPGS, p.o.) or MK-1064 and vehicle (saline and 8% DMSO, i.p.) or CNO 90 min prior to the start of the 30-min restraint. This timing was chosen based on the fact that both MK-1064 and CNO have been shown to promote behavioral effects in the rat within 30 min of administration and effects last up to 4 h after administration (Alexander et al., 2009 (link); Farrell and Roth, 2013 (link); Hasegawa et al., 2014 (link); Roecker et al., 2014 (link)). Additionally, CNO was given at the time of MK-1064 injection to minimize the effects of repeated handling prior to restraint.

GRAFE L.A., EACRET D., LUZ S., GOTTER A.L., RENGER J.J., WINROW C.J, & BHATNAGAR S. (2017). OREXIN 2 RECEPTOR REGULATION OF THE HYPOTHALAMIC–PITUITARY–ADRENAL (HPA) RESPONSE TO ACUTE AND REPEATED STRESS. Neuroscience, 348, 313-323.

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Anesthesia and Pharmacological Reagents

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Isoflurane used in the study was obtained from RWD Life Science (Shenzhen, China). Lidocaine and pentobarbital were purchased from Chaohui Pharmaceutical (Shanghai, China). Clozapine N-oxide (CNO) was purchased from Sigma-Aldrich (United States, C0832).

Bu X., Chen Y., Lv P., Fu X, & Fu B. (2022). Glutamatergic neurons in paraventricular nucleus of the thalamus regulate the recovery from isoflurane anesthesia. BMC Anesthesiology, 22, 256.

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Fluoxetine Treatment Behavioral Effects

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Behavioral tests were conducted by a researcher unaware of the genotype, as previously described [5 (link)]. Chronic treatment consisted of 18 days of free drinking of fluoxetine (0.167 mg/ml)/saccharine (1%) mixture or saccharine alone (as vehicle). Acute fluoxetine (7.5 mg/kg) or saline (as vehicle) was administered intraperitoneally, 15 min before the test. For chemogenetic studies, one dose of clozapine N-oxide (4 mg/kg in saline, Sigma Aldrich, C0832) was injected intraperitoneally 30 min before the test.

Sagi Y., Medrihan L., George K., Barney M., McCabe K.A, & Greengard P. (2019). Emergence of 5-HT5A signaling in parvalbumin neurons mediates delayed antidepressant action. Molecular Psychiatry, 25(6), 1191-1201.

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DREADD-Mediated Neuronal Modulation in Animals

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For neuronal modulation of animals expressing DREADDs, Clozapine N-oxide (Sigma, 2.5mg/kg) was diluted in physiological 1xPBS and injected intraperitoneally 30 min before the start of the experimental session. Animals that received this treatment in experiments were habituated by PBS injections during handling sessions.

Grössl F., Munsch T., Meis S., Griessner J., Kaczanowska J., Pliota P., Kargl D., Badurek S., Kraitsy K., Rassoulpour A., Zuber J., Lessmann V, & Haubensak W. (2018). Dorsal tegmental dopamine neurons gate associative learning of fear. Nature neuroscience, 21(7), 952-962.

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Clozapine-N-Oxide Administration Protocol

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Clozapine-N-Oxide was purchased from Sigma-Aldrich, St. Louis, MO, United States For in vivo experiments, CNO was dissolved into sterile saline at a concentration of 0.5 mg/mL, and was sterile filtered prior to injection. Injections were given intraperitoneal (i.p.) in a volume of 50–250 μl per mg of mouse weight 30–45 min prior to seizure induction by audio or HC stimulation. For in vitro experiments, CNO was dissolved into dimethyl sulfoxide (DMSO) at a concentration of 10 mM and diluted 10,000-fold into ACSF for experiments.

Wenker I.C., Boscia A.R., Lewis C., Tariq A., Miralles R., Hanflink J.C., Saraf P, & Patel M.K. (2022). Forebrain epileptiform activity is not required for seizure-induced apnea in a mouse model of Scn8a epilepsy. Frontiers in Neural Circuits, 16, 1002013.

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Pharmacological Manipulation of Rodents

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Clozapine-N-Oxide (Sigma) was diluted in sterile saline and administered at 1 mg/kg i.p. 30 min prior to behavioral testing. Istradefylline (Tocris # 5417) was dissolved in DMSO and diluted to 10% DMSO, 1% NP-40 in sterile saline immediately prior to i.p. administration at 3 mg/kg. Velneperit (MEdChem Express #342577-38-2) has very low solubility in water, thus it was dissolved in pure DMSO prior to injection and injected at 100 mg/kg in 0.03 ml using an insulin syringe. Bexarotene (Tocris # 5819) also has limited solubility in water, thus it was dissolved in pure DMSO prior to injection and injected i.p. at 50 mg/kg in a volume of 0.03 ml using an insulin syringe. Control animals received equal volumes of vehicle. These volumes of pure DMSO have been previously tested and validated to cause no adverse health effects in adult mice.

McCullough K.M., Daskalakis N.P., Gafford G., Morrison F.G, & Ressler K.J. (2018). Cell-type-specific interrogation of CeA Drd2 neurons to identify targets for pharmacological modulation of fear extinction. Translational Psychiatry, 8, 164.

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