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

Abstract

Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N -desmethylclozapine ( N -Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long–Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d -amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d -amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N -Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N -Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

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References 63

1. 1.

   Agulhon C, Boyt KM, Xie AX, Friocourt F, Roth BL, McCarthy KD (2013 ) Modulation of the autonomic nervous system and behaviour by acute glial cell Gq protein-coupled receptor activation in vivo. J Physiol 591:5599–5609.

   Not in our library10.1113/jphysiol.2013.261289

2. 2.

   Alexander GM, Rogan SC, Abbas AI, Armbruster BN, Pei Y, Allen JA, Nonneman RJ, Hartmann J, Moy SS, Nicolelis MA, McNamara JO, Roth BL (2009 ) Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63:27–39.

   Not in our library10.1016/j.neuron.2009.06.014

3. 3.

   Alves-Rodrigues A, Leurs R, Willems E, Timmerman H (1996 ) Binding of clozapine metabolites and analogues to the histamine H3 receptor in rat brain cortex. Arch Pharm (Weinheim) 329:413–416.

   Not in our library10.1002/ardp.19963290808

4. 4.

   Anderson SA, Michaelides M, Zarnegar P, Ren Y, Fagergren P, Thanos PK, Wang GJ, Bannon M, Neumaier JF, Keller E, Volkow ND, Hurd YL (2013 ) Impaired periamygdaloid-cortex prodynorphin is characteristic of opiate addiction and depression. J Clin Invest 123:5334–5341.

   Not in our library10.1172/jci70395

5. 5.

   Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL (2007 ) Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A 104:5163–5168.

   Not in our library10.1073/pnas.0700293104

6. 6.

   Baldessarini RJ, Centorrino F, Flood JG, Volpicelli SA, Huston-Lyons D, Cohen BM (1993 ) Tissue concentrations of clozapine and its metabolites in the rat. Neuropsychopharmacology 9:117–124.

   Not in our library10.1038/npp.1993.50

7. 7.

   Boender AJ, de Jong JW, Boekhoudt L, Luijendijk MC, van der Plasse G, Adan RA (2014 ) Combined use of the canine adenovirus-2 and DREADD-technology to activate specific neural pathways in vivo. PLoS One 9:e95392.

   Not in our library10.1371/journal.pone.0095392

8. 8.

   Bull C, Freitas KC, Zou S, Poland RS, Syed WA, Urban DJ, Minter SC, Shelton KL, Hauser KF, Negus SS, Knapp PE, Bowers MS (2014 ) Rat nucleus accumbens core astrocytes modulate reward and the motivation to self-administer ethanol after abstinence. Neuropsychopharmacology 39:2835–2845.

   Not in our library10.1038/npp.2014.135

9. 9.

   Bun H, Disdier B, Aubert C, Catalin J (1999 ) Interspecies variability and drug interactions of clozapine metabolism by microsomes. Fundam Clin Pharmacol 13:577–581.

   Not in our library10.1111/j.1472-8206.1999.tb00364.x

10. 10.

    Chang SE, Todd TP, Bucci DJ, Smith KS (2015 ) Chemogenetic manipulation of ventral pallidal neurons impairs acquisition of sign-tracking in rats. Eur J Neurosci 42:3105–3116.

    Not in our library10.1111/ejn.13103

11. 11.

    Chang WH, Lin SK, Lane HY, Wei FC, Hu WH, Lam YW, Jann MW (1998 ) Reversible metabolism of clozapine and clozapine N-oxide in schizophrenic patients. Prog Neuropsychopharmacol Biol Psychiatry 22:723–739.

    Not in our library10.1016/s0278-5846(98)00035-9

12. 12.

    Chen X, Choo H, Huang XP, Yang X, Stone O, Roth BL, Jin J (2015 ) The first structure-activity relationship studies for designer receptors exclusively activated by designer drugs. ACS Chem Neurosci 6:476–484.

    Not in our library10.1021/cn500325v

13. 13.

    Conti LH, Costill JE, Flynn S, Tayler JE (2005 ) Effects of a typical and an atypical antipsychotic on the disruption of prepulse inhibition caused by corticotropin-releasing factor and by rat strain. Behav Neurosci 119:1052–1060.

    Not in our library10.1037/0735-7044.119.4.1052

14. 14.

    Coward DM (1992 ) General pharmacology of clozapine. Br J Psychiatry Suppl (17):5–11.

    Not in our library

15. 15.
    Controlling signaling with a specifically designed Gi-coupled receptor · 1998

    Not checked
16. 16.

    Cremers TI, Flik G, Hofland C, Stratford RE Jr. (2012 ) Microdialysis evaluation of clozapine and N-desmethylclozapine pharmacokinetics in rat brain. Drug Metab Dispos 40:1909–1916.

    Not in our library10.1124/dmd.112.045682

17. 17.

    Davies MA, Compton-Toth BA, Hufeisen SJ, Meltzer HY, Roth BL (2005 ) The highly efficacious actions of N-desmethylclozapine at muscarinic receptors are unique and not a common property of either typical or atypical antipsychotic drugs: is M1 agonism a pre-requisite for mimicking clozapine's actions?. Psychopharmacology (Berl) 178:451–460.

    Not in our library10.1007/s00213-004-2017-1

18. 18.

    Dell’Anno MT, Caiazzo M, Leo D, Dvoretskova E, Medrihan L, Colasante G, Giannelli S, Theka I, Russo G, Mus L, Pezzoli G, Gainetdinov RR, Benfenati F, Taverna S, Dityatev A, Broccoli V (2014 ) Remote control of induced dopaminergic neurons in parkinsonian rats. J Clin Invest 124:3215–3229.

    Not in our library10.1172/jci74664

19. 19.

    España RA, Oleson EB, Locke JL, Brookshire BR, Roberts DC, Jones SR (2010 ) The hypocretin-orexin system regulates cocaine self-administration via actions on the mesolimbic dopamine system. Eur J Neurosci 31:336–348.

    Not in our library10.1111/j.1460-9568.2009.07065.x

20. 20.

    España RA, Melchior JR, Roberts DC, Jones SR (2011 ) Hypocretin 1/orexin A in the ventral tegmental area enhances dopamine responses to cocaine and promotes cocaine self-administration. Psychopharmacology 214:415–426.

    Not in our library10.1007/s00213-010-2048-8

21. 21.

    Farrell MS, Pei Y, Wan Y, Yadav PN, Daigle TL, Urban DJ, Lee HM, Sciaky N, Simmons A, Nonneman RJ, Huang XP, Hufeisen SJ, Guettier JM, Moy SS, Wess J, Caron MG, Calakos N, Roth BL (2013 ) A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons. Neuropsychopharmacology 38:854–862.

    Not in our library10.1038/npp.2012.251

22. 22.

    Feifel D, Shilling PD, Melendez G (2011 ) Clozapine and PD149163 elevate prepulse inhibition in Brown Norway rats. Behav Neurosci 125:268–272.

    Not in our library10.1037/a0022691

23. 23.

    Ferguson SM, Eskenazi D, Ishikawa M, Wanat MJ, Phillips PE, Dong Y, Roth BL, Neumaier JF (2011 ) Transient neuronal inhibition reveals opposing roles of indirect and direct pathways in sensitization. Nat Neurosci 14:22–24.

    Not in our library10.1038/nn.2703

24. 24.

    Ferguson SM, Phillips PE, Roth BL, Wess J, Neumaier JF (2013 ) Direct-pathway striatal neurons regulate the retention of decision-making strategies. J Neurosci 33:11668–11676.

    Not in our library10.1523/jneurosci.4783-12.2013

25. 25.
    Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs for use in wild-type animals · 2015

    Not checked
26. 26.

    Grace PM, Strand KA, Galer EL, Urban DJ, Wang X, Baratta MV, Fabisiak TJ, Anderson ND, Cheng K, Greene LI, Berkelhammer D, Zhang Y, Ellis AL, Yin HH, Campeau S, Rice KC, Roth BL, Maier SF, Watkins LR (2016 ) Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation. Proc Natl Acad Sci U S A 113:E3441–E3450.

    Not in our library10.1073/pnas.1602070113

27. 27.

    Guettier JM, Gautam D, Scarselli M, Ruiz de Azua I, Li JH, Rosemond E, Ma X, Gonzalez FJ, Armbruster BN, Lu H, Roth BL, Wess J (2009 ) A chemical-genetic approach to study G protein regulation of beta cell function in vivo. Proc Natl Acad Sci U S A 106:19197–19202.

    Not in our library10.1073/pnas.0906593106

28. 28.

    Huang XF, Tan YY, Huang X, Wang Q (2007 ) Effect of chronic treatment with clozapine and haloperidol on 5-HT(2A and 2C) receptor mRNA expression in the rat brain. Neurosci Res 59:314–321.

    Not in our library10.1016/j.neures.2007.08.001

29. 29.

    Jann MW, Grimsley SR, Gray EC, Chang WH (1993 ) Pharmacokinetics and pharmacodynamics of clozapine. Clin Pharmacokinet 24:161–176.

    Not in our library10.2165/00003088-199324020-00005

30. 30.

    Jann MW, Lam YW, Chang WH (1994 ) Rapid formation of clozapine in guinea-pigs and man following clozapine-N-oxide administration. Arch Int Pharmacodyn Ther 328:243–250.

    Not in our library

31. 31.
    Chemical–genetic attenuation of focal neocortical seizures · 2014

    No notice
32. 32.

    Keith VA, Mansbach RS, Geyer MA (1991 ) Failure of haloperidol to block the effects of phencyclidine and dizocilpine on prepulse inhibition of startle. Biol Psychiatry 30:557–566.

    Not in our library10.1016/0006-3223(91)90025-h

33. 33.

    Lin G, McKay G, Hubbard JW, Midha KK (1994 ) Decomposition of clozapine N-oxide in the qualitative and quantitative analysis of clozapine and its metabolites. J Pharm Sci 83:1412–1417.

    Not in our library10.1002/jps.2600831010

34. 34.

    Lin G, McKay G, Midha KK (1996 ) Characterization of metabolites of clozapine N-oxide in the rat by micro-column high performance liquid chromatography/mass spectrometry with electrospray interface. J Pharm Biomed Anal 14:1561–1577.

    Not in our library10.1016/0731-7085(96)01738-4

35. 35.

    Ma S, Allocca G, Ong-Palsson EK, Singleton CE, Hawkes D, McDougall SJ, Williams SJ, Bathgate RA, Gundlach AL (2016 ) Nucleus incertus promotes cortical desynchronization and behavioral arousal. Brain Struct Funct. Advance online publication. Retrieved October 13, 2016. doi:10.1007/s00429-016-1230-0.

    Not in our library10.1007/s00429-016-1230-0

36. 36.

    Marchant NJ, Whitaker LR, Bossert JM, Harvey BK, Hope BT, Kaganoversusky K, Adhikary S, Prisinzano TE, Vardy E, Roth BL, Shaham Y (2016 ) Behavioral and physiological effects of a novel kappa-opioid receptor-based DREADD in rats. Neuropsychopharmacology 41:402–409.

    Not in our library10.1038/npp.2015.149

37. 37.

    Michaelides M, Anderson SA, Ananth M, Smirnov D, Thanos PK, Neumaier JF, Wang GJ, Volkow ND, Hurd YL (2013 ) Whole-brain circuit dissection in free-moving animals reveals cell-specific mesocorticolimbic networks. J Clin Invest 123:5342–5350.

    Not in our library10.1172/jci72117

38. 38.

    Mizoguchi H, Katahira K, Inutsuka A, Fukumoto K, Nakamura A, Wang T, Nagai T, Sato J, Sawada M, Ohira H, Yamanaka A, Yamada K (2015 ) Insular neural system controls decision-making in healthy and methamphetamine-treated rats. Proc Natl Acad Sci U S A 112:E3930–E3939.

    Not in our library10.1073/pnas.1418014112

39. 39.

    Mosier KE, Song J, McKay G, Hubbard JW, Fang J (2003 ) Determination of clozapine, and its metabolites, N-desmethylclozapine and clozapine N-oxide in dog plasma using high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 783:377–382.

    Not in our library10.1016/s1570-0232(02)00655-4

40. 40.

    Natesan S, Reckless GE, Barlow KB, Nobrega JN, Kapur S (2007 ) Evaluation of N-desmethylclozapine as a potential antipsychotic–preclinical studies. Neuropsychopharmacology 32:1540–1549.

    Not in our library10.1038/sj.npp.1301279

41. 41.

    Peters J-U (2012 ) Polypharmacology in drug discovery. Hoboken, NJ: Wiley.

    Not in our library

42. 42.

    Pienaar IS, Gartside SE, Sharma P, De Paola V, Gretenkord S, Withers D, Elson JL, Dexter DT (2015 ) Pharmacogenetic stimulation of cholinergic pedunculopontine neurons reverses motor deficits in a rat model of Parkinson's disease. Mol Neurodegener 10:47

    Not in our library10.1186/s13024-015-0044-5

43. 43.

    Pirmohamed M, Williams D, Madden S, Templeton E, Park BK (1995 ) Metabolism and bioactivation of clozapine by human liver in vitro. J Pharmacol Exp Ther 272:984–990.

    Not in our library

44. 44.

    Qiu MH, Chen MC, Fuller PM, Lu J (2016 ) Stimulation of the pontine parabrachial nucleus promotes wakefulness via extra-thalamic forebrain circuit nodes. Curr Biol 26:2301–2312.

    Not in our library10.1016/j.cub.2016.07.054

45. 45.

    Ray RS, Corcoran AE, Brust RD, Kim JC, Richerson GB, Nattie E, Dymecki SM (2011 ) Impaired respiratory and body temperature control upon acute serotonergic neuron inhibition. Science 333:637–642.

    Not in our library10.1126/science.1205295

46. 46.

    Redfern CH, Coward P, Degtyarev MY, Lee EK, Kwa AT, Hennighausen L, Bujard H, Fishman GI, Conklin BR (1999 ) Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nat Biotechnol 17:165–169.

    Not in our library10.1038/6165

47. 47.

    Robinson S, Todd TP, Pasternak AR, Luikart BW, Skelton PD, Urban DJ, Bucci DJ (2014 ) Chemogenetic silencing of neurons in retrosplenial cortex disrupts sensory preconditioning. J Neurosci 34:10982–10988.

    Not in our library10.1523/jneurosci.1349-14.2014

48. 48.

    Salmi P, Ahlenius S (1996 ) Further evidence for clozapine as a dopamine D1 receptor agonist. Eur J Pharmacol 307:27–31.

    Not in our library10.1016/0014-2999(96)00181-1

49. 49.

    Schotte A, Janssen PF, Megens AA, Leysen JE (1993 ) Occupancy of central neurotransmitter receptors by risperidone, clozapine and haloperidol, measured ex vivo by quantitative autoradiography. Brain Res 631:191–202.

    Not in our library10.1016/0006-8993(93)91535-z

50. 50.

    Scofield MD, Boger HA, Smith RJ, Li H, Haydon PG, Kalivas PW (2015 ) Gq-DREADD selectively initiates glial glutamate release and inhibits cue-induced cocaine seeking. Biol Psychiatry 78:441–451.

    Not in our library10.1016/j.biopsych.2015.02.016

51. 51.

    Sengupta A, Winters B, Bagley EE, McNally GP (2016 ) Disrupted prediction error links excessive amygdala activation to excessive fear. J Neurosci 36:385–395.

    Not in our library10.1523/jneurosci.3670-15.2016

52. 52.

    Sur C, Mallorga PJ, Wittmann M, Jacobson MA, Pascarella D, Williams JB, Brandish PE, Pettibone DJ, Scolnick EM, Conn PJ (2003 ) N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-D-aspartate receptor activity. Proc Natl Acad Sci U S A 100:13674–13679.

    Not in our library10.1073/pnas.1835612100

53. 53.

    Swerdlow NR, Bakshi V, Geyer MA (1996 ) Seroquel restores sensorimotor gating in phencyclidine-treated rats. J Pharmacol Exp Ther 279:1290–1299.

    Not in our library10.1016/0924-977x(96)87794-7

54. 54.

    Swerdlow NR, Weber M, Qu Y, Light GA, Braff DL (2008 ) Realistic expectations of prepulse inhibition in translational models for schizophrenia research. Psychopharmacology (Berl) 199:331–388.

    Not in our library10.1007/s00213-008-1072-4

55. 55.

    Thomas DR, Dada A, Jones GA, Deisz RA, Gigout S, Langmead CJ, Werry TD, Hendry N, Hagan JJ, Davies CH, Watson JM (2010 ) N-desmethylclozapine (NDMC) is an antagonist at the human native muscarinic M(1) receptor. Neuropharmacology 58:1206–1214.

    Not in our library10.1016/j.neuropharm.2010.02.017

56. 56.

    Wang S, Tan Y, Zhang JE, Luo M (2013 ) Pharmacogenetic activation of midbrain dopaminergic neurons induces hyperactivity. Neurosci Bull 29:517–524.

    Not in our library10.1007/s12264-013-1327-x

57. 57.

    Wicker E, Forcelli PA (2016 ) Chemogenetic silencing of the midline and intralaminar thalamus blocks amygdala-kindled seizures. Exp Neurol 283:404–412.

    Not in our library10.1016/j.expneurol.2016.07.003

58. 58.

    Wirtshafter D, Stratford TR (2016 ) Chemogenetic inhibition of cells in the paramedian midbrain tegmentum increases locomotor activity in rats. Brain Res 1632:98–106.

    Not in our library10.1016/j.brainres.2015.12.014

59. 59.

    Witten IB, Steinberg EE, Lee SY, Davidson TJ, Zalocusky KA, Brodsky M, Yizhar O, Cho SL, Gong S, Ramakrishnan C, Stuber GD, Tye KM, Janak PH, Deisseroth K (2011 ) Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement. Neuron 72:721–733.

    Not in our library10.1016/j.neuron.2011.10.028

60. 60.

    Wong G, Kuoppamäki M, Hietala J, Lüddens H, Syvälahti E, Korpi ER (1996 ) Effects of clozapine metabolites and chronic clozapine treatment on rat brain GABAA receptors. Eur J Pharmacol 314:319–323.

    Not in our library10.1016/s0014-2999(96)00671-1

61. 61.

    Yau JO, McNally GP (2015 ) Pharmacogenetic excitation of dorsomedial prefrontal cortex restores fear prediction error. J Neurosci 35:74–83.

    Not in our library10.1523/jneurosci.3777-14.2015

62. 62.

    Yorgason JT, España RA, Jones SR (2011 ) Demon voltammetry and analysis software: analysis of cocaine-induced alterations in dopamine signaling using multiple kinetic measures. J Neurosci Methods 202:158–164.

    Not in our library10.1016/j.jneumeth.2011.03.001

63. 63.

    Zhu H, Pleil KE, Urban DJ, Moy SS, Kash TL, Roth BL (2014 ) Chemogenetic inactivation of ventral hippocampal glutamatergic neurons disrupts consolidation of contextual fear memory. Neuropsychopharmacology 39:1880–1892.

    Not in our library10.1038/npp.2014.35

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