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Review Article

Pro-psychotic effects of synthetic cannabinoids: interactions with central dopamine, serotonin, and glutamate systems

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Pages 65-73 | Received 17 Dec 2017, Accepted 11 Jan 2018, Published online: 31 Jan 2018

References

  • Abi-Dargham A, Laruelle M, Aghajanian GK, Charney D, Krystal J. 1997. The role of serotonin in the pathophysiology and treatment of schizophrenia. J Neuropsychiatry Clin Neurosci. 9:1–17.
  • Benford DM, Caplan JP. 2011. Psychiatric sequelae of Spice, K2, and synthetic cannabinoid receptor agonists. Psychosomatics. 52:295.
  • Brakoulias V. 2012. Products containing synthetic cannabinoids and psychosis. Aust N Z J Psychiatry. 46:281–282.
  • Braun M. 1960. Reserpine as a therapeutic agent in schizophrenia. Am J Psychiatry. 116:744–5.
  • Brents LK, Reichard EE, Zimmerman SM, Moran JH, Fantegrossi WE, Prather PL. 2011. Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity. PLoS One. 6:e21917.
  • Brown TM, Brotchie JM, Fitzjohn SM. 2003. Cannabinoids decrease corticostriatal synaptic transmission via an effect on glutamate uptake. J Neurosci. 23:11073–7.
  • Bubar MJ, Cunningham KA. 2006. Serotonin 5-HT2A and 5-HT2C receptors as potential targets for modulation of psychostimulant use and dependence. Curr Top Med Chem. 6:1971–1985.
  • Caballero A, Tseng KY. 2012. Association of cannabis use during adolescence, prefrontal CB1 receptor signaling, and schizophrenia. Front Pharmacol. 3:101.
  • Canazza I, Ossato A, Trapella C, Fantinati A, De Luca MA, Margiani G, Vincenzi F, Rimondo C, Di Rosa F, Gregori A, et al. 2016. Effect of the novel synthetic cannabinoids AKB48 and 5F-AKB48 on “tetrad, sensorimotor, neurological and neurochemical responses in mice. In vitro and in vivo pharmacological studies”. Psychopharmacology (Berl). 233:3685–3709.
  • Carhart-Harris R, Nutt D. 2017. Serotonin and brain function: a tale of two receptors. J Psychopharmacol). 31:1091–1120.
  • Carlsson A, Lindquist M. 1963. Effect of chlorpromazine or haloperidol on formation of 3methoxytyramine and normetanephrine in mouse brain. Acta Pharmacol Toxicol (Copenh). 20:140–144.
  • Celada P, Puig MV, Amargós-Bosch M, Adell A, Artigas F. 2004. The therapeutic role of 5-HT1A and 5-HT2A receptors in depression. J Psychiatry Neurosci. 29:252–265.
  • Cohen BD, Rosenbaum G, Luby ED, Gottlieb JS. 1962. Comparison of phencyclidine hydrochloride (Sernyl) with other drugs. Simulation of schizophrenic performance with phencyclidine hydrochloride (Sernyl), lysergic acid diethylamide (LSD-25), and amobarbital (Amytal) sodium; II. Symbolic and sequential thinking. Arch Gen Psychiatry. 6:395–401.
  • Compton DR, Johnson MR, Melvin LS, Martin BR. 1992. Pharmacological profile of a series of bicyclic cannabinoid analogs: classification as cannabimimetic agents. J Pharmacol Exp Ther. 260:201–209.
  • Darmani NA, Pandya DK. 2000. Involvement of other neurotransmitters in behaviors induced by the cannabinoid CB1 receptor antagonist SR 141716A in naive mice. J Neural Transm. 107:931–945.
  • Darmani NA. 2001. Cannabinoids of diverse structure inhibit two DOI-induced 5-HT2A receptor-mediated behaviors in mice. Pharmcol Biochem Behav. 68:311–317.
  • Davies BM, Beech HR. 1960. The effect of 1-arylcylohexylamine (sernyl) on twelve normal volunteers. J Ment Sci. 106:912–924.
  • De Almeida J, Palacios JM, Mengood G. 2008. Distribution of 5-HT and DA receptors in primate prefrontal cortex: implications for pathophysiology and treatment. Prog Brain Res. 172:101–115.
  • Demuth DG, Molleman A. 2006. Cannabinoid signalling. Life Sci. 78:549–563.
  • Derkinderen P, Valjent E, Toutant M, Corvol JC, Enslen H, Ledent C, Trzaskos J, Caboche J, Girault JA. 2003. Regulation of extracellular signal-regulated kinase by cannabinoids in hippocampus. J Neurosci. 23:2371–82.
  • Den Boon FS, Chameau P, Schaafsma-Zhao Q, van Aken W, Bari M, Oddi S, Werkman TR. 2012. Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors. Proc Natl Acad Sci USA. 109:3534–3539.
  • D’Souza DC, Sewell RA, Ranganathan M. 2009. Cannabis and psychosis/schizophrenia: human studies. Eur Arch Psychiatry Clin Neurosci. 259:413–431.
  • Durand D, Delgado LL, de la Parra-Pellot DM, Nichols-Vinueza D. 2013. Psychosis and severe rhabdomyolysis associated with synthetic cannabinoid use. Clin Schizophr Relat Psychoses. 8:205–208.
  • Egashira N, Shirakawa A, Okuno R, Mishima K, Iwasaki K, Oishi R, Fujiwara M. 2011. Role of endocannabinoid and glutamatergic systems in DOI-induced head-twitch response in mice. Pharmacol Biochem Behav. 99:52–58.
  • Eggan SM, Lewis DA. 2007. Immunocytochemical distribution of the cannabinoid Cb1 receptor in the primate neocortex: a regional and laminr analysis. Cereb Cortex. 17:119–175.
  • El Khoury MA, Gorgievski V, Moutsimilli L, Giros B, Tzavara ET. 2012. Interactions between the cannabinoid and dopaminergic systems: evidence from animal studies. Prog Neuropsychopharmacol Biol Psychiatry. 38:36–50.
  • Elmore JS, Bauman MH. 2017. Serotonergic responsiveness after repeated exposure to the synthetic cannabinoid JWH-018. Drug Alcohol Depend. 171:e59.
  • Every-Palmer S. 2010. Warning: legal synthetic cannabinoid-receptor agonists such as JWH-018 may precipitate psychosis in vulnerable individuals. Addiction. 105:1859–1860.
  • Every-Palmer S. 2011. Synthetic cannabinoid JWH-018 and psychosis: an explorative study. Drug Alcohol Depend. 117:152–157.
  • Evins AE, Green AI, Kane JM, Murray RM. 2012. The effect of marijuana use on the risk for schizophrenia. J Clin Psychiatry. 73:1463–1468.
  • Feigenbaum JJ, Bergmann F, Richmond SA, Mechoulam R, Nadler V, Kloog Y, Sokolovsky M. 1989. Nonpsychotropic cannabinoid acts as a functional N-methyl-D-aspartate receptor blocker. Proc Natl Acad Sci USA. 86:9584–7.
  • Fergusson DM, Poulton R, Smith PF, Boden JM. 2006. Drugs: cannabis and psychosis. BMJ. 332:172–175.
  • Ford BM, Tai S, Fantegrossi WE, Prather PL. 2017. Synthetic pot: not your Grandfather’s Marijuana. Trends Pharmacol Sci. 38:257–276.
  • Franklin JM, Carrasco GA. 2012. Cannabinoid-induced enhanced interaction and protein levels of serotonin 5-HT2A and dopamine D2Receptors in rat prefrontal cortex. J Psychopharmacol. 26:1333–1347.
  • Franklin JM, Mathew M, Carrasco GA. 2013. Cannabinoid-induced upregulation of serotonin 2A receptors in the hypothalamic paraventricular nucleus and anxiety-like behaviors in rats. Neurosci Lett. 548:165–169.
  • Gatch MB, Forster MJ. 2014. Δ9-Tetrahydrocannabinol-like effects of novel synthetic cannabinoids found on the Gray Market. Behav Pharmacol. 26:460–468.
  • Gatch MB, Forster MJ. 2016. Δ9-Tetrahydrocannabinol-like effects of novel synthetic cannabinoids in mice and rats. Psychopharmacology (Berlin). 233:1901–1910.
  • Gessa GL, Casu MA, Carta G, Mascia MS. 1998. Cannabinoids decrease acetylcholine release in the medial-prefrontal cortex and hippocampus, reversal by SR 141716A. Eur J Pharmacol. 355:119–24.
  • Glue P, Al-Shaqsi S, Hancock D, Gale C, Strong B, Schep L. 2013. Hospitalisation associated with use of the synthetic cannabinoid K2. NZ Med J. 126:18–23.
  • Gonzalez-Maeso J, Sealfon SC. 2009. Psychedelics and schizophrenia. Trends Neurosci. 32:225–232.
  • Gore CP, Egan GP, Walton D. 1957. The place of reserpine in the treatment of the chronic psychotic patient. Am J Psychiatry. 114:333–7.
  • Gray JA, Roth BL. 2001. Paradoxical trafficking and regulation of 5-HT(2A) receptors by agonists and antagonists. Brain Res Bull. 56:441–445.
  • Hampson RE, Miller F, Palchik G, Deadwyler SA. 2011. Cannabinoid receptor activation modifies NMDA receptor mediated release of intracellular calcium: implications for endocannabinoid control of hippocampal neural plasticity. Neuropharmacology. 60:944–52.
  • Hashimotodani Y, Ohno-Shosaku T, Watanabe M, Kano M. 2007. Roles of phospholipase Cbeta and NMDA receptor in activity-dependent endocannabinoid release. J Physiol. 584:373–80.
  • Hermanns-Clausen M, Kneisel S, Szabo B, Auwärter V. 2013. Acute toxicity due to the confirmed consumption of synthetic cannabinoids: clinical and laboratory findings. Addiction. 108:534–544.
  • Higley JD, Linnoila M. 1997. Low central nervous system serotonergic activity is traitlike and correlates with impulsive behavior. A nonhuman primate model investigating genetic and environmental influences on neurotransmission. Ann NY Acad Sci. 836:39–56.
  • Hoffman AF, Lupica CR. 2000. Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. J Neurosci. 20:2470–2479.
  • Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Porrino LJ. 2014. Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology. 47 Suppl 1:345–58.
  • Hurst D, Loeffler G, McLay R. 2011. Psychosis associated with synthetic cannabinoid agonists: a case series. Am J Psychiatry. 168:1119.
  • Janoyan JJ, Crim JL, Darmani NA. 2002. Reversal of SR 141716A-induced head-twitch and ear-scratch responses in mice by Δ9-THC and other cannabinoids. Pharmcol Biochem Behav. 71:155–162.
  • Jones KA, Srivastava DP, Allen JA, Strachan RT, Roth BL, Penzes P. 2009. Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling. Proc Natl Acad Sci. USA. 106:19575–19580.
  • Kearn CS, Blake-Palmer K, Daniel E, Mackie K, Glass M. 2005. Concurrent stimulation of Cannabinoid CB1 and dopamine D2 receptors enhances heterodimer formation: a mechanism for receptor cross-talk? Mol Pharmacol. 67:1697–1704.
  • Khaspekov LG, Brenz Verca MS, Frumkina LE, Hermann H, Marsicano G, Lutz B. 2004. Involvement of brain-derived neurotrophic factor in cannabinoid receptor-dependent protection against excitotoxicity. Eur J Neurosci. 19:1691–8.
  • Kim SH, Won SJ, Mao XO, Jin K, Greenberg DA. 2006. Molecular mechanisms of cannabinoid protection from neuronal excitotoxicity. Mol Pharmacol. 69:691–6.
  • Kim J, Li Y. 2015. Chronic activation of CB2 cannabinoid receptors in the hippocampus increases excitatory synaptic transmission. J Physiol. 593:871–886.
  • Kölfavi A, Rodrigues RJ, Ledent C, Mackie K, Vizi ES, Cunha RA, Sperlágh B. 2005. Involvement of cannabinoid receptors in the regulation of neurotransmitter release in the rodent striatum: a combined immunochemical and pharmacological analysis. J Neurosci. 25:2874–2884.
  • Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, Heninger GR, Bowers MB Jr, Charney DS. 1994. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry. 51:199–214.
  • Li Q, Yan H, Wilson WA, Swartzwelder HS. 2010 Modulation of NMDA and AMPA-mediated synaptic transmission by CB1 receptors in frontal cortical pyramidal cells. Brain Res. 1342:127–37.
  • Liu Q, Bhat M, Bowen WD and Cheng J. 2009. Signaling pathways from cannabinoid receptor-1 activation to inhibition of N-methyl-D-aspartic acid mediated calcium influx and neurotoxicity in dorsal root ganglion neurons. J Pharmacol Exp Ther. 331:1062–70.
  • Maksymetz J, Moran SP and Conn PJ. 2017. Targeting metabotropic glutamate receptors for novel treatments of schizophrenia. Mol Brain. 10(1):15.
  • Malhotra AK, Pinals DA, Weingartner H, Sirocco K, Missar CD, Pickar D, Breier A. 1996. NMDA receptor function and human cognition: the effects of ketamine in healthy volunteers. Neuropsychopharmacology. 14:301–307.
  • Marek GJ, Martin-Ruiz R, Abo A, Artigas F. 2005. The selective 5-HT2A receptor antagonist M100907 enhances antidepressant-like behavioral effects of the SSRI fluoxetine. Neuropsychopharmacology. 30:2205–2215.
  • Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, Azad SC, Cascio MG, Gutiérrez SO, van der Stelt M, et al. 2003. CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science. 302:84–8.
  • Martin WR, Wikler A, Eades CG, Pescor FT. 1963. Tolerance to and physical dependence on morphine in rats. Psychopharmacologia. 4:247–260.
  • Maeng S, Zarate CA Jr. 2007. The role of glutamate in mood disorders: results from the ketamine in major depression study and the presumed cellular mechanism underlying its antidepressant effects. Curr Psychiatry Rep. 9:467–74.
  • Meltzer HY, Matsubara S, Lee JC. 1989. Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-1, D-2 and serotonin2 pKi values. J Pharmacol Exp Ther. 251:238–46.
  • Meltzer HY. 1991. The mechanism of action of novel antipsychotic drugs. Schizophr Bull. 17:263–87.
  • Meltzer HY. 1999. The role of serotonin in antipsychotic drug action. Neuropsychopharmacology. 21:106S–115S.
  • Meltzer HY, Li Z, Kaneda Y, Ichikawa J. 2003. Serotonin receptors: their key role in drugs to treat schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry. 27: 1159–1172.
  • Melis M, Pistis M, Perra S, Muntoni AL, Pillolla G, Gessa GL (2004) Endocannabinoids mediate presynaptic inhibition of glutamatergic transmission in rat ventral tegmental area dopamine neurons through activation of CB1 receptors. J Neurosci. 24:53–62.
  • Monte AA, Calello DP, Gerona RR, Hamad E, Campleman SL, Brent J, Wax P, ACMT Toxicology Investigators Consortium (ToxIC). 2017. Characteristics and treatment of patients with clinical illness due to synthetic cannabinoid inhalation reported by medical toxicologists: a toxic database study. J Med Toxicol. 13:146–152.
  • Morera-Herreras T, Ruiz-Ortega JA, Gómez-Urquijo S, Ugedo L. 2008. Involvement of subthalamic nucleus in the stimulatory effect of Delta(9)-tetrahydrocannabinol on dopaminergic neurons. Neuroscience. 151:817–823.
  • Müller H, Sperling W, Köhrmann M, Huttner HB, Kornhuber J, Maler JM. 2010. The synthetic cannabinoid Spice as a trigger for an acute exacerbation of cannabis induced recurrent psychotic episodes. Schizophr Res. 118:309–310.
  • Munro S, Thomas KL, Abu-Shaar M. 1993. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 365:61–65.
  • National Institutes of Health (US); Biological Sciences Curriculum Study. NIH Curriculum Supplement Series [Internet]. Bethesda (MD): National Institutes of Health (US); 2007. Information about Mental Illness and the Brain. https://www.ncbi.nlm.nih.gov/books/NBK20369/
  • Navarrete F, Rodríguez-Arias M, Martín-García E, Navarro D, García-Gutiérrez MS, Aguilar MA, Manzanares J. 2013. Role of CB2 cannabinoid receptors in the rewarding, reinforcing, and physical effects of nicotine. Neuropsychopharmacology. 38:2515–2524.
  • Nichols DE. 2004. Hallucinogens. Pharmacol Ther. 101:131–181.
  • Oluwabusi OO, Lobach L, Akhtar U, Youngman B, Ambrosini PJ. 2013. Synthetic cannabinoid-induced psychosis: two adolescent cases. J Child Adolesc Psychopharmacol. 22:393–395.
  • Onaivi ES, Ishiguro H, Gong JP, Patel S, Meozzi PA, Myers L, Perchuk A, Mora Z, Tagliaferro PA, Gardner E. et al. 2008. Brain neuronal CB2 cannabinoid receptors in drug abuse and depression: from mice to human subjects. PLoS One. 3:e1640.
  • Ossato A, Uccelli L, Bilel S, Canazza I, Di Domenico G, Pasquali M, Pupillo G, De Luca MA, Boschi A, Vincenzi F, et al. 2017. Psychostimulant effect of the synthetic cannabinoid JWH-018 and AKB48: behavioral, neurochemical, and dopamine transporter scan imaging studies in mice. Front Psychiatry. 8:1–13.
  • Peglow S, Buchner J, Briscoe G. 2012. Synthetic cannabinoid induced psychosis in a previously nonpsychotic patient. Am J Addict. 21:287–288.
  • Pertwee RG. 2006. Cannabinoid pharmacology: the first 66 years. Brit J Pharmacol. 147(Suppl 1);S163–S171.
  • Raote I, Bhattacharya A, Panicker MM. 2007. Serotonin 2A(5-HT2A) Receptor Function: Ligand-Dependent Mechanisms and Pathways. In: Chattopadhyay A, editor. Serotonin Receptors in Neurobiology. Boca Raton (FL): CRC Press/Taylor & Francis (Chapter 6).
  • Rapport MM, Green AA, Page IH (1948) Serum vasoconstrictor, serotonin; isolation and characterization. J Biol Chem. 176:1243–51.
  • Rapport MM. 1949. Serum vasoconstrictor (serotonin) the presence of creatinine in the complex; a proposed structure of the vasoconstrictor principle. J Biol Chem. 180:961–9.
  • Reggio PH. 2010. Endocannabinoid binding to the cannabinoid receptors: what is known and what remains unknown. CMC. 17:1468–1486.
  • Roberto AJ, Lorenzo A, Li KJ, Young J, Mohan A, Pinnaka S, Lapidus KAB. 2016. First-episode of synthetic cannabinoid-induced psychosis in a young adult, successfully managed with hospitalization and risperidone. Case Rep Psychiatry. 2016:7257489.
  • Rodriguez JS, McMahon LR. 2014. JWH-018 in rhesus monkeys: differential antagonism of discriminative stimulus, rate-decreasing, and hypothermic effects. Eur J Pharmacol. 740:151–9.
  • Rogoz Z. 2013. Combined treatment with atypical antipsychotics and antidepressants in treatment-resistant depression: preclinical and clinical efficacy. Pharmacol Rep. 65:1535–1544.
  • Rubino T, Parolaro D. 2013. Cannabis abuse in adolescence and the risk of psychosis: a brief review of the preclinical evidence. Prog Neuropsychopharmacol Biol Psychiatry. 52:41–44.
  • Sánchez-Blázquez P, Rodríguez-Muñoz M, Garzón J. 2014. The cannabinoid receptor 1 associates with NMDA receptors to produce glutamatergic hypofunction: implications in psychosis and schizophrenia. Front Pharmacol. 4:169.
  • Sawa A, Snyder SH. 2003. Schizophrenia: neural mechanisms for novel therapies. Mol Med. 9:3–9.
  • Seely KA, Lapoint J, Moran JH, Fattore L. 2012. Spice drugs are more than harmless herbal blends: a review of the pharmacology and toxicology of synthetic cannabinoids. Prog Neuropsychopharmacol Biol Psychiatry. 39:234–243.
  • Seeman P. 2002. Atypical antipsychotics: mechanism of action. Can J Psychiatry. 47:27–38.
  • Spaderna M, Addy PH, D’Souza DC. 2013. Spicing things up: synthetic cannabinoids. Psychopharmacology (Berlin). 228:525–540.
  • Tai S, Hyatt WS, Gu C, Franks LN, Vasiljevik T, Brents LK, Prather PL, Fantegrossi WE. 2015. Repeated administration of phytocannabinoid Δ(9)-THC or synthetic cannabinoids JWH-018 and JWH-073 induces tolerance to hypothermia but not locomotor suppression in mice, and reduces CB1 receptor expression and function in a brain region-specific manner. Pharmacol Res. 102:22–32.
  • Tai S, Fantegrossi WE. 2017. Pharmacological and toxicological effects of synthetic cannabinoids and their metabolites. Curr Top Behav Neurosci. 32:249–262.
  • Tanda G, Pontieri FE, Di Chiara G. 1997. Cannabinoid and heroin activation of mesolimbic dopamine transmission by a common MU1 opioid receptor mechanism. Science. 276:2048–2050.
  • Tsou K, Mackie K, Sanudo-Pena MC, Walker JM. 1999. Cannabinoid CB1receptors are localized primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation. Neuroscience. 93:969–975.
  • Tsuang HC, Chen WJ, Lin SH, Chen TY, Chang YL, Huang KH, Lane HY. 2013. Impaired impulse control is associated with a 5-HT2A receptor polymorphism in schizophrenia. Psychiatry Res. 208:105–110.
  • Tung CK, Chiang TP, Lam M. 2012. Acute mental disturbance caused by synthetic cannabinoid: a potential emerging substance of abuse in Hong Kong. East Asian Arch Psychiatry. 22:31–33.
  • Vallersnes OM, Dines AM, Wood DM, Yates C, Heyerdahl F, Hovda KE, Giraudon I, Euro-DEN Research Group, 2016. Psychosis associated with acute recreational drug toxicity: a European case series. BMC Psychiatry. 16:293.
  • van Amsterdam J, Brunt T, van den Brink W (2015) The adverse health effects of synthetic cannabinoids with emphasis on psychosis-like effects. J Psychopharmacol. 29:254–63.
  • Volkow ND, Hampson AJ, Baler RD. 2017. Don’t worry, be happy: endocannabinoids and cannabis at the intersection of stress and reward. Annu Rev Pharmacol Toxicol. 57:285–308.
  • Wang S, Sun H, Liu S, Wang T, Guan J, Jia J. 2016. Role of hypothalamic cannabinoid receptors in post-stroke depression in rats. Brain Res Bull. 121:91–97.
  • Woolley DW, Shaw E. 1954. A biochemical and pharmacological suggestion about certain mental disorders. Proc Natl Acad Sci USA. 40:228–31.
  • Wright K, Rooney N, Feeney M, Tate J, Robertson D, Welham M, Ward S. 2005. Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing. Gastroenterology. 129:437–453.

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