Effects of positive mGlu5 modulation on D 2 signaling and nicotine-conditioned place preference: Mechanisms of epigenetic inheritance in a transgenerational model of drug abuse vulnerability in psychosis
Restricted accessResearch articleFirst published online March, 2025
Effects of positive mGlu5 modulation on D 2 signaling and nicotine-conditioned place preference: Mechanisms of epigenetic inheritance in a transgenerational model of drug abuse vulnerability in psychosis
The metabotropic glutamate type 5 (mGlu5) receptor has emerged as a potential target for the treatment of psychosis that is suggested to have greater efficacy than antipsychotic medications that are currently utilized.
Aims:
This study sought to elucidate mechanisms of therapeutic action associated with the modulation of the mGlu5 receptor in a disordered system marked by dopamine dysfunction. We further explored epigenetic mechanisms contributing to heritable transmission of a psychosis-like phenotype in a novel heritable model of drug abuse vulnerability in psychosis.
Methods:
F1 generation male and female Sprague-Dawley rats that were the offspring of two neonatal quinpirole-treated (QQ) or two saline-treated (SS) animals were tested on nicotine-conditioned place preference (CPP). Regulators of G protein signaling 9 (RGS9) and β-arrestin 2 (βA2), which mediate dopamine (DA) D2 signaling, were measured in the nucleus accumbens shell, prelimbic and infralimbic cortices. Reduced Representation Bisulfite Sequencing (RRBS) was used to analyze the cytosine methylation in these brain regions.
Results:
Pretreatment with the mGlu5-positive allosteric modulator 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) 20 min prior to conditioning trials blocked enhanced nicotine CPP and mitigated aberrant G protein-dependent and -independent signaling in QQ animals. RRBS analysis revealed region-specific changes in several pathways, including nicotine addiction, dopamine synapses, and neural connectivity.
Conclusions:
These results reveal an important region-specific mechanism of action for CDPPB in a system marked by enhanced DAD2 receptor signaling. Results additionally reveal DNA methylation as an epigenetic mechanism of heritability, further validating the current model as a useful tool for the study of psychosis and comorbid nicotine use.
AgnatiLFGuidolinDAlbertinG, et al (2010) An integrated view on the role of receptor mosaics at perisynaptic level: Focus on adenosine A2A, dopamine D2, cannabinoid CB1, and metabotropic glutamate mGlu5 receptors. J Recept Signal Trans30: 355–369.
2.
AkalinAKormakssonMLiS, et al. (2012) methylKit: A comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol13: R87.
3.
ArsovaAMøllerTCHellyerSD, et al (2021) Positive allosteric modulators of metabotropic glutamate receptor 5 as tool compounds to study signaling bias. Mol Pharmacol99: 328–341.
4.
BatelP (2000) Addiction and schizophrenia. Eur Psychiatry15: 115–122.
5.
BeaulieuJ-MGainetdinovRR (2011) The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev63: 182–217.
6.
BeaulieuJ-MSotnikovaTDMarionS, et al (2005) An Akt/β-Arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell122: 261–273.
7.
BeggiatoSTomasiniMCBorelliAC, et al (2016) Functional role of striatal A2A, D2, and mGlu5 receptor interactions in regulating striatopallidal GABA neuronal transmission. J Neurochem138: 254–264.
8.
BonciAHopfFW (2005) The dopamine D2 receptor: New surprises from an old friend. Neuron47: 335–338.
9.
BørglumADDemontisDGroveJ, et al (2014) Genome-wide study of association and interaction with maternal cytomegalovirus infection suggests new schizophrenia loci. Mol Psychiatry19: 325–333.
10.
BrownRWPernaMKNoelDM, et al (2011) Amphetamine locomotor sensitization and conditioned place preference in adolescent male and female rats neonatally treated with quinpirole. Behav Pharmacol22: 374–378.
11.
BrownRWMapleAMPernaMK, et al (2012) Schizophrenia and substance abuse comorbidity: Nicotine addiction and the neonatal quinpirole model. Develop Neurosci34: 140–151.
12.
BrownRWKirbySLDentonAR, et al (2018) An analysis of the rewarding and aversive associative properties of nicotine in the neonatal quinpirole model: Effects on glial cell line-derived neurotrophic factor (GDNF). Schizophr Res194: 107–114.
13.
BrownRWVarnumCGWillsLJ, et al (2021) Modulation of mGlu5 improves sensorimotor gating deficits in rats neonatally treated with quinpirole through changes in dopamine D2 signaling. Pharmacol Biochem Behav211: 173292.
14.
CabelloNGandíaJBertarelliDCG, et al (2009) Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher-order oligomers in living cells. J Neurochem109: 1497–1507.
15.
ChavesCDursunSMTusconiM, et al (2024) Neuroinflammation and schizophrenia—is there a link?Front Psychiatry15: 1356975.
16.
CopeZAHugginsKNSheppardAB, et al (2010) Neonatal quinpirole treatment enhances locomotor activation and dopamine release in the nucleus accumbens core in response to amphetamine treatment in adulthood. Synapse64: 289–300.
17.
CorrellJANoelDMSheppardAB, et al (2009) Nicotine sensitization and analysis of brain-derived neurotrophic factor in adolescent ß-arrestin-2 knockout mice. Synapse63: 510–519.
18.
CosfordNDPTehraniLRoppeJ, et al (2003) 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]- pyridine: A potent and highly selective metabotropic glutamate subtype 5 receptor antagonist with anxiolytic activity. J Med Chem46: 204–206.
19.
CzamaraDDieckmannLLahti-PulkkinenM, et al (2024) Sex differences in DNA methylation across gestation: A large scale, cross-cohort, multi-tissue analysis. Cell Mol Life Sci81: 177.
20.
De LeonJDiazFJ (2005) A meta-analysis of worldwide studies demonstrates an association between schizophrenia and tobacco smoking behaviors. Schizophr Res76: 135–157.
21.
Del’GuidiceTLemassonMBeaulieuJ-M (2011) Role of beta-Arrestin 2 downstream of dopamine receptors in the basal ganglia. Front Neuroanatomy5: 58.
22.
DograSConnPJ (2022) Metabotropic glutamate receptors as emerging targets for the treatment of schizophrenia. Mol Pharmacol101: 275–285.
23.
EllwoodITPatelTWadiaV, et al (2017) Tonic or phasic stimulation of dopaminergic projections to prefrontal cortex causes mice to maintain or deviate from previously learned behavioral strategies. J Neurosci37: 8315–8329.
24.
ErmakovEAMelamudMMBunevaVN, et al (2022) Immune system abnormalities in schizophrenia: An integrative view and translational perspectives. Front Psychiatry13: 880568.
25.
FerreSvon EulerGJohanssonB, et al (1991) Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes. Proc Natl Acad Sci U S A88: 7238–7241.
26.
FinotelloFCamilloBD (2015) Measuring differential gene expression with RNA-seq: Challenges and strategies for data analysis. Brief Funct Genom14: 130–142.
27.
FöckingMDoyleBMunawarN, et al (2019) Epigenetic factors in schizophrenia: Mechanisms and experimental approaches. Mol Neuropsychiatry5: 6–12.
28.
FromerMRoussosPSiebertsSK, et al (2016) Gene expression elucidates functional impact of polygenic risk for schizophrenia. Nat Neurosci19: 1442–1453.
29.
FultzEKQuadirSGMartinD, et al (2021) ERK-directed phosphorylation of mGlu5 gates methamphetamine reward and reinforcement in mouse. Int J Mol Sci22: 1473.
30.
GejmanPVSandersARDuanJ (2010) The role of genetics in the etiology of schizophrenia. Psychiatr Clin North Am33: 35–66.
31.
GillWDSheltonHWBurgessKC, et al (2020) Effects of an adenosine A2A agonist on the rewarding associative properties of nicotine and neural plasticity in a rodent model of schizophrenia. J Psychopharmacol34: 137–144.
32.
GillWDBurgessKCViedC, et al (2021) Transgenerational evidence of increases in dopamine D2 receptor sensitivity in rodents: Impact on sensorimotor gating, the behavioral response to nicotine and BDNF. J Psychopharmacol35: 1188–1203.
33.
GobinCSchwendtM (2020) The cognitive cost of reducing relapse to cocaine-seeking with mGlu5 allosteric modulators. Psychopharmacology237: 115–125.
34.
GogtayNVyasNSTestaR, et al (2011) Age of onset of schizophrenia: Perspectives from structural neuroimaging studies. Schizophr Bull37: 504–513.
GraceAA (1991) Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: A hypothesis for the etiology of schizophrenia. Neuroscience41: 1–24.
37.
GraybielAM (2000) The basal ganglia. Curr Biol10: R509–R511.
38.
GurevichVVGurevichEV (2020) Biased GPCR signaling: Possible mechanisms and inherent limitations. Pharmacol Ther211: 107540.
39.
GusevAMancusoNWonH, et al (2018) Transcriptome-wide association study of schizophrenia and chromatin activity yields mechanistic disease insights. Nat Genet50: 538–548.
40.
HallFSDrgonovaJJainS, et al (2013) Implications of genome wide association studies for addiction: Are our a priori assumptions all wrong?Pharmacol Ther140: 267–279.
41.
HámorPUGobinCMSchwendtM (2020) The role of glutamate mGlu5 and adenosine A2a receptor interactions in regulating working memory performance and persistent cocaine seeking in rats. Prog Neuropsychopharmacol Biol Psychiatry103: 109979.
42.
HawiZTongJDarkC, et al (2018) The role of cadherin genes in five major psychiatric disorders: A literature update. Am J Med Genet B Neuropsychiatr Genet177: 168–180.
43.
HiranoSTakeichiM (2012) Cadherins in brain morphogenesis and wiring. Physiol Rev92: 597–634.
44.
HuT-MChenS-JHsuS-H, et al (2019) Functional analyses and effect of DNA methylation on the EGR1 gene in patients with schizophrenia. Psychiatry Res275: 276–282.
45.
HuYFangZYangY, et al (2018) Analyzing the genes related to nicotine addiction or schizophrenia via a pathway and network based approach. Scient Rep8: 2894.
46.
HuiMBeierKT (2022) Defining the interconnectivity of the medial prefrontal cortex and ventral midbrain. Front Mol Neurosci15: 971349.
47.
IwamotoKBundoMYamadaK, et al (2005) DNA methylation status of SOX10 correlates with its downregulation and oligodendrocyte dysfunction in schizophrenia. J Neurosci25: 5376–5381.
48.
JaffeAEStraubREShinJH, et al (2018) Developmental and genetic regulation of the human cortex transcriptome illuminate schizophrenia pathogenesis. Nat Neurosci21: 1117–1125.
49.
JangHSShinWJLeeJE, et al (2017) CpG and non-CpG methylation in epigenetic gene regulation and brain function. Genes8: 148.
50.
JanicijevicSMDejanovicSDBorovcaninM (2020) Interplay of brain-derived neurotrophic factor and cytokines in schizophrenia. Serb J Exp Clin Res21: 283–289.
51.
JonesPATakaiD (2001) The role of DNA methylation in mammalian epigenetics. Science293: 1068–1070.
52.
JouvertPRevelM-OLazarisA, et al (2004) Activation of the cGMP pathway in dopaminergic structures reduces cocaine-induced EGR-1 expression and locomotor activity. J Neurosci24: 10716–10725.
53.
KaplanGXuHAbreuK, et al (2022) DNA epigenetics in addiction susceptibility. Front Genet13: 806685.
54.
KhlghatyanJQuintanaCParentM, et al (2019) High sensitivity mapping of cortical dopamine D2 receptor expressing neurons. Cerebral Cortex29: 3813–3827.
55.
KimK-M (2023) Unveiling the differences in signaling and regulatory mechanisms between dopamine D2 and D3 receptors and their impact on behavioral sensitization. Int J Mol Sci24: 6742.
56.
KinneyGGO’BrienJALemaireW, et al (2005) A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models. J Pharmacol Exp Ther313: 199–206.
57.
KoobGFVolkowND (2010) Neurocircuitry of addiction. Neuropsychopharmacology35: 217–238.
58.
KostrzewaRMNowakPBrusR, et al (2016) Perinatal treatments with the dopamine D2-receptor agonist quinpirole produces permanent D2-receptor supersensitization: A model of schizophrenia. Neurochem Res41: 183–192.
59.
KostrzewaRMWydraKFilipM, et al (2018) Dopamine D2 receptor supersensitivity as a spectrum of neurotoxicity and status in psychiatric disorders. J Pharmacol Exp Ther366: 519–526.
60.
KotsiriIRestaPSpyrantisA, et al (2023) Viral infections and schizophrenia: A comprehensive review. Viruses15: 1345.
61.
KrokenRASommerIESteenVM, et al (2019) Constructing the immune signature of schizophrenia for clinical use and research; An integrative review translating descriptives into diagnostics. Front Psychiatry9: 753.
62.
LeaPMFadenAI (2006) Metabotropic glutamate receptor subtype 5 antagonists MPEP and MTEP. CNS Drug Rev12: 149–166.
63.
LeonardSFreedmanR (2006) Genetics of chromosome 15q13-q14 in schizophrenia. Biol Psychiatry60: 115–122.
64.
LiebermanJAStroupTSMcEvoyJP, et al (2005) Effectiveness of antipsychotic drugs in patients with chronic schizophrenia. N Engl J Med353: 1209–1223.
65.
LiouYChenMWangY, et al (2009) Analysis of genetic variations in the RGS9 gene and antipsychotic-induced tardive dyskinesia in schizophrenia. Am J Med Genet B Neuropsychiatr Genet150B: 239–242.
66.
LoscalzoJHandyDE (2013) Epigenetic modifications: Basic mechanisms and role in cardiovascular disease (2013 Grover Conference Series). Pulm Circ4: 169–174.
67.
MacDonaldJLRoskamsAJ (2009) Epigenetic regulation of nervous system development by DNA methylation and histone deacetylation. Prog Neurobiol88: 170–183.
68.
MagwaiTShangaseKBOgingaFO, et al (2021) DNA methylation and schizophrenia: Current literature and future perspective. Cells10: 2890.
69.
MapleAMSmithKJPernaMK, et al (2015) Neonatal quinpirole treatment produces prepulse inhibition deficits in adult male and female rats. Pharmacol Biochem Behav137: 93–100.
70.
Marszalek-GrabskaMGawelKMatosiukD, et al (2020) Effects of the positive allosteric modulator of metabotropic glutamate receptor 5, VU-29, on maintenance association between environmental cues and rewarding properties of ethanol in rats. Biomolecules10: 793.
71.
MasriBSalahpourADidriksenM, et al (2008) Antagonism of dopamine D2 receptor/β-arrestin 2 interaction is a common property of clinically effective antipsychotics. Proc Natl Acad Sci105: 13656–13661.
72.
McCarthyMMNugentBMLenzKM (2017) Neuroimmunology and neuroepigenetics in the establishment of sex differences in the brain. Nat Rev Neurosci18: 471–484.
73.
McCutcheonRAAbi-DarghamAHowesOD (2019) Schizophrenia, dopamine and the striatum: From biology to symptoms. Trends Neurosci42: 205–220.
74.
McGeehanAJOliveMF (2003) The mGluR5 antagonist MPEP reduces the conditioned rewarding effects of cocaine but not other drugs of abuse. Synapse47: 240–242.
75.
MenaARuiz-SalasJCPuentesA, et al (2016) Reduced prepulse inhibition as a biomarker of schizophrenia. Front Behav Neurosci10: 202.
76.
MendizabalIBertoSUsuiN, et al (2019) Cell type-specific epigenetic links to schizophrenia risk in the brain. Genome Biol20: 135.
77.
MiyamotoSDuncanGEMarxCE, et al (2005) Treatments for schizophrenia: A critical review of pharmacology and mechanisms of action of antipsychotic drugs. Mol Psychiatry10: 79–104.
78.
MuellerDBravo-RiveraCQuirkGJ (2010) Infralimbic D2 receptors are necessary for fear extinction and extinction-related tone responses. Biol Psychiatry68: 1055–1060.
79.
NishiokaMBundoMKasaiK, et al (2012) DNA methylation in schizophrenia: progress and challenges of epigenetic studies. Genome Medicine4(12): 96.
80.
OdaYKanaharaNIyoM (2015) Alterations of dopamine D2 receptors and related receptor-interacting proteins in schizophrenia: The pivotal position of dopamine supersensitivity psychosis in treatment-resistant schizophrenia. Int J Mol Sci16: 30144–30163.
81.
OtaKTPierreVJPloskiJE, et al (2008) The NO-cGMP-PKG signaling pathway regulates synaptic plasticity and fear memory consolidation in the lateral amygdala via activation of ERK/MAP kinase. Learn Memory15: 792–805.
82.
OtsukaIWatanabeYHishimotoA, et al (2015) Association analysis of the Cadherin13 gene with schizophrenia in the Japanese population. Neuropsychiatr Dis Treat11: 1381–1393.
83.
PatersonNESemenovaSGaspariniF, et al (2003) The mGluR5 antagonist MPEP decreased nicotine self-administration in rats and mice. Psychopharmacology167: 257–264.
84.
PeetersLDWillsLJCuozzoAM, et al (2023) Reinstatement of nicotine conditioned place preference in a transgenerational model of drug abuse vulnerability in psychosis: Impact of BDNF on the saliency of drug associations. Psychopharmacology240: 1453–1464.
85.
PeetersLDWillsLJCuozzoAM, et al (2024) Modulation of mGlu5 reduces rewarding associative properties of nicotine via changes in mesolimbic plasticity: Relevance to comorbid cigarette smoking in psychosis. Pharmacol Biochem Behav239: 173752.
86.
PernaMKBrownRW (2013) Adolescent nicotine sensitization and effects of nicoctine on accumbal dopamine release in a rodent model of increased dopamine D2 recepetor sensitivity. Behav Brain Res242: 102–109.
87.
PetersonDJGillWDDoseJM, et al. (2017) The effects of nicotine in the neonatal quinpirole rodent model of psychosis: Neural plasticity mechanisms and nicotinic receptor changes. Behav Brain Res325: 17–24.
88.
PopoliPPèzzolaATorvinenM, et al (2001) The selective mGlu5 receptor agonist CHPG inhibits quinpirole-induced turning in 6-hydroxydopamine-lesioned rats and modulates the binding characteristics of dopamine D2 receptors in the rat striatum interactions with adenosine A2a receptors. Neuropsychopharmacology25: 505–513.
89.
Porter-StranskyKAWeinshenkerD (2017) Arresting the development of addiction: The role of β-Arrestin2 in drug abuse. J Pharmacol Exp Ther361: 341–348.
90.
PowellSBZhouXGeyerMA (2009) Prepulse inhibition and genetic mouse models of schizophrenia. Behav Brain Res204: 282–294.
91.
RahmanZSchwarzJGoldSJ, et al (2003) RGS9 modulates dopamine signaling in the basal ganglia. Neuron38: 941–952.
92.
RediesCHertelNHübnerCA (2012) Cadherins and neuropsychiatric disorders. Brain Res1470: 130–144.
93.
RiveroGGabilondoAMGarcía-FusterMJ, et al (2012) Differential regulation of RGS proteins in the prefrontal cortex of short- and long-term human opiate abusers. Neuropharmacology62: 1044–1051.
94.
RiveroOSichSPoppS, et al (2013) Impact of the ADHD-susceptibility gene CDH13 on development and function of brain networks. Eur Neuropsychopharmacol23: 492–507.
95.
RiveroOSeltenMMSichS, et al (2015) Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition. Transl Psychiatry5: e655.
96.
RuttenKvan der KamELVryJD, et al (2011) The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) potentiates conditioned place preference induced by various addictive and non-addictive drugs in rats. Addict Biol16: 108–115.
97.
SaklothFPolizuCBertheratF, et al (2020) Regulators of G protein signaling in analgesia and addiction. Mol Pharmacol98: 739–750.
98.
SeemanMVSeemanP (2014) Is schizophrenia a dopamine supersensitivity psychotic reaction?Prog Neuropsychopharmacol Biol Psychiatry48: 155–160.
99.
SeemanPKoFJackE, et al (2007) Consistent with dopamine supersensitivity, RGS9 expression is diminished in the amphetamine-treated animal model of schizophrenia and in postmortem schizophrenia brain. Synapse61: 303–309.
100.
ShimSShumanMDuncanE (2016) An emerging role of cGMP in the treatment of schizophrenia: A review. Schizophr Res170: 226–231.
101.
SmithJSRajagopalS (2016) The β-Arrestins: Multifunctional regulators of G protein-coupled receptors. J Biol Chem291: 8969–8977.
102.
SozuguzelMDSazciAYildizM (2019) Female gender specific association of the Reelin (RELN) gene rs7341475 variant with schizophrenia. Mol Biol Rep46: 3411–3416.
103.
SunDMaJZPayneTJ, et al (2008) β-Arrestins 1 and 2 are associated with nicotine dependence in European American smokers. Mol Psychiatry13: 398–406.
104.
SwerdlowNRBraffDLGeyerMA (1990) GABAergic projection from nucleus accumbers to ventral pallidum mediates dopamine-induced sensorimotor gating deficits of acoustic startle in rats. Brain Res532: 146–150.
105.
TraynorJRTerziDCaldaroneBJ, et al (2009) RGS9-2: Probing an intracellular modulator of behavior as a drug target. Trends Pharmacol Sci30: 105–111.
106.
UrsNMSnyderJCJacobsenJPR, et al (2012) Deletion of GSK3β in D2R-expressing neurons reveals distinct roles for β-arrestin signaling in antipsychotic and lithium action. Proc Natl Acad Sci109: 20732–20737.
107.
ValléeA (2022) Neuroinflammation in schizophrenia: The key role of the WNT/β-catenin pathway. Int J Mol Sci23: 2810.
108.
van NimwegenLde HaanLvan BeverenN, et al (2005) Adolescence, schizophrenia and drug abuse: A window of vulnerability. Acta Psychiatr Scand111: 35–42.
109.
VolkowNLiT-K (2005) The neuroscience of addiction. Nat Neurosci8: 1429–1430.
110.
VolkowNDWangG-JFowlerJS, et al (2012) Addiction circuitry in the human brain. Ann Rev Pharmacol Toxicol52: 321–336.
111.
VriesLDZhengBFischerT, et al (2000) The regulator of G protein signaling family. Pharmacol Toxicol40: 235–271.
112.
WardasJ (2008) Potential role of adenosine A2A receptors in the treatment of schizophrenia. Front Biosci13: 4071.
113.
WidholmJJGassJTClevaRM, et al (2012) The Mglur5 positive allosteric modulator CDPPB does not alter extinction or contextual reinstatement of methamphetamine-seeking behavior in rats. J Addict Res Ther S1: 004.
114.
XiaYDaiRWangK, et al (2021) Sex-differential DNA methylation and associated regulation networks in human brain implicated in the sex-biased risks of psychiatric disorders. Mol Psychiatry26: 835–848.
115.
XieWBarrCLKimA, et al (2012) Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome. Cell148: 816–831.
116.
XuHWangFLiuY, et al (2014) Sex-biased methylome and transcriptome in human prefrontal cortex. Human Mol Genet23: 1260–1270.
117.
YararbasGKeserAKanitL, et al (2010) Nicotine-induced conditioned place preference in rats: Sex differences and the role of mGluR5 receptors. Neuropharmacology58: 374–382.
118.
ZachariouVGeorgescuDSanchezN, et al (2003) Essential role for RGS9 in opiate action. Proc Natl Acad Sci100: 13656–13661.
119.
ZhouJXiaYLiM, et al (2023) A higher dysregulation burden of brain DNA methylation in female patients implicated in the sex bias of schizophrenia. Mol Psychiatry28: 4842–4852.
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