Molecular Biology Reports

, Volume 46, Issue 3, pp 2867–2875 | Cite as

Correlation between the epigenetic modification of histone H3K9 acetylation of NR2B gene promoter in rat hippocampus and ethanol withdrawal syndrome

  • Duan Li
  • Yanqing Zhang
  • Yanting Zhang
  • Qi Wang
  • Qin Miao
  • Yahui Xu
  • Jair C. Soares
  • Xiangyang ZhangEmail author
  • Ruiling ZhangEmail author
Original Article


Patients with alcohol use disorder may develop acute ethanol withdrawal syndrome (EWS). Previous studies showed that an epigenetic modification of the N-methyl-d-aspartate (NMDA) receptor, especially NMDA receptor 2B subunit (NR2B), was involved in the pathological process of EWS. However, the relationship between the epigenetic regulation of the NR2B gene in the rat hippocampus region and EWS were inconsistent. The purpose of this study was to explore the role of the histone H3K9 acetylation of the NR2B gene in the rat hippocampus region in EWS. A rat model of chronic ethanol exposure was established. EWS score and the behavioral changes were recorded at different time points. The NR2B expression levels and the histone H3K9 acetylation level in the NR2B gene promoter region were measured using qRT-PCR, Western blot, immunofluorescence, and chromatin immunoprecipitation, respectively. Finally, the relationship between the epigenetic modification of histone H3K9 acetylation of NR2B gene promoter and EWS were examined. Our ultimate results showed that the EWS score was increased at 2 h, peaked at 6 h after withdrawal of ethanol, and reduced to the level parallel to the normal control group at day 3 after ethanol withdrawal. The NR2B mRNA expression and protein levels showed similar patterns. Further correlation analyses indicted that both histone H3K9 acetylation in NR2B gene promoter and the expression levels of NR2B were positively associated with EWS. Our results suggest that chronic ethanol exposure may result in epigenetic modification of histone H3K9 acetylation in NR2B gene promoter in rat hippocampus, and the expression levels of NR2B were found to be positively correlated with ethanol withdrawal syndrome.


Chronic ethanol exposure Ethanol withdrawal syndrome Epigenetic modification N-Methyl-d-aspartate receptor N-Methyl-d-aspartate receptor 2B subunit (NR2B) 



Ethanol withdrawal syndrome




N-Methyl-d-aspartate receptor 2 B


Chromatin immunoprecipitation


Quantitative real-time PCR


Chronic intermittent ethanol



This work was supported by the National Natural Science Foundation of China (No. 81471351), Open Program of Henan Key Laboratory of Biological Psychiatry (No. ZDSYS2016007) and the Programs for Science and Technology Development of Henan (No. 172102310499).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11033_2019_4733_MOESM1_ESM.docx (208 kb)
Supplementary material 1 (DOCX 207 KB)


  1. 1.
    Crews F, He J, Hodge C (2007) Adolescent cortical development: a critical period of vulnerability for addiction. Pharmacol Biochem Behav 86(2):189–199. CrossRefGoogle Scholar
  2. 2.
    Stehman CR, Mycyk MB (2013) A rational approach to the treatment of alcohol withdrawal in the ED. Am J Emerg Med 31(4):734–742. CrossRefGoogle Scholar
  3. 3.
    Perry EC (2014) Inpatient management of acute alcohol withdrawal syndrome. CNS Drugs 28(5):401–410. CrossRefGoogle Scholar
  4. 4.
    Awissi DK, Lebrun G, Fagnan M, Skrobik Y (2013) Alcohol, nicotine, and iatrogenic withdrawals in the ICU. Crit Care Med 41(9 Suppl 1):S57–S68. CrossRefGoogle Scholar
  5. 5.
    CP. OB (1996) Drug addiction and drug abuse. Goodman and Gilman’s the pharmacological basis of therapeutics. McGraw-Hill, New YorkGoogle Scholar
  6. 6.
    Thompson WL (1978) Management of alcohol withdrawal syndromes. Arch InterN Med 138(2):278–283CrossRefGoogle Scholar
  7. 7.
    Uzbay IT, Erden BF, Tapanyigit EE, Kayaalp SO (1997) Nitric oxide synthase inhibition attenuates signs of ethanol withdrawal in rats. Life Sci 61(22):2197–2209CrossRefGoogle Scholar
  8. 8.
    Dodd PR, Foley PF, Buckley ST, Eckert AL, Innes DJ (2004) Genes and gene expression in the brain of the alcoholic. Addict Behav 29(7):1295–1309. CrossRefGoogle Scholar
  9. 9.
    Carlson RW, Kumar NN, Wong-Mckinstry E, Ayyagari S, Puri N, Jackson FK, Shashikumar S (2012) Alcohol withdrawal syndrome. Crit Care Clin 28(4):549–585. CrossRefGoogle Scholar
  10. 10.
    Kaniakova M, Krausova B, Vyklicky V, Korinek M, Lichnerova K, Vyklicky L, Horak M (2012) Key amino acid residues within the third membrane domains of NR1 and NR2 subunits contribute to the regulation of the surface delivery of N-methyl-D-aspartate receptors. J Biol Chem 287(31):26423–26434. CrossRefGoogle Scholar
  11. 11.
    Paoletti P, Bellone C, Zhou Q (2013) NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 14(6):383–400. CrossRefGoogle Scholar
  12. 12.
    Liu H, Wang H, Sheng M, Jan LY, Jan YN, Basbaum AI (1994) Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn. Proc Natl Acad Sci USA 91(18):8383–8387CrossRefGoogle Scholar
  13. 13.
    Lett TA, Voineskos AN, Kennedy JL, Levine B, Daskalakis ZJ (2014) Treating working memory deficits in schizophrenia: a review of the neurobiology. Biol Psychiatry 75(5):361–370. CrossRefGoogle Scholar
  14. 14.
    Self DW, Nestler EJ (1995) Molecular mechanisms of drug reinforcement and addiction. Annu Rev Neurosci 18:463–495. CrossRefGoogle Scholar
  15. 15.
    Maler JM, Esselmann H, Wiltfang J, Kunz N, Lewczuk P, Reulbach U, Bleich S, Ruther E, Kornhuber J (2005) Memantine inhibits ethanol-induced NMDA receptor up-regulation in rat hippocampal neurons. Brain Res 1052(2):156–162. CrossRefGoogle Scholar
  16. 16.
    Ryabinin AE (1998) Role of hippocampus in alcohol-induced memory impairment: implications from behavioral and immediate early gene studies. Psychopharmacology 139(1–2):34–43CrossRefGoogle Scholar
  17. 17.
    Goebel DJ, Poosch MS (1999) NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A. Brain Res Mol Brain Res 69(2):164–170CrossRefGoogle Scholar
  18. 18.
    Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12(3):529–540CrossRefGoogle Scholar
  19. 19.
    Fadda F, Rossetti ZL (1998) Chronic ethanol consumption: from neuroadaptation to neurodegeneration. Prog Neurobiol 56(4):385–431CrossRefGoogle Scholar
  20. 20.
    Jacobson RR, Acker CF, Lishman WA (1990) Patterns of neuropsychological deficit in alcoholic Korsakoff’s syndrome. Psychol Med 20(2):321–334CrossRefGoogle Scholar
  21. 21.
    Pian JP, Criado JR, Milner R, Ehlers CL (2010) N-methyl-D-aspartate receptor subunit expression in adult and adolescent brain following chronic ethanol exposure. Neuroscience 170(2):645–654. CrossRefGoogle Scholar
  22. 22.
    Warnault V, Darcq E, Levine A, Barak S, Ron D (2013) Chromatin remodeling—a novel strategy to control excessive alcohol drinking. Transl Psychiatry 3:e231. CrossRefGoogle Scholar
  23. 23.
    Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447(7143):407–412. CrossRefGoogle Scholar
  24. 24.
    Pandey SC, Ugale R, Zhang H, Tang L, Prakash A (2008) Brain chromatin remodeling: a novel mechanism of alcoholism. J Neurosci 28(14):3729–3737. CrossRefGoogle Scholar
  25. 25.
    Moonat S, Sakharkar AJ, Zhang H, Tang L, Pandey SC (2013) Aberrant histone deacetylase2-mediated histone modifications and synaptic plasticity in the amygdala predisposes to anxiety and alcoholism. Biol Psychiatry 73(8):763–773. CrossRefGoogle Scholar
  26. 26.
    Peterson CL, Laniel MA (2004) Histones and histone modifications. Curr Biol 14(14):R546–R551. CrossRefGoogle Scholar
  27. 27.
    Qiang M, Denny A, Lieu M, Carreon S, Li J (2011) Histone H3K9 modifications are a local chromatin event involved in ethanol-induced neuroadaptation of the NR2B gene. Epigenetics 6(9):1095–1104. CrossRefGoogle Scholar
  28. 28.
    Qiang M, Denny A, Chen J, Ticku MK, Yan B, Henderson G (2010) The site specific demethylation in the 5′-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription. PLoS ONE 5(1):e8798. CrossRefGoogle Scholar
  29. 29.
    Erden BF, Ozdemirci S, Yildiran G, Utkan T, Gacar N, Ulak G (1999) Dextromethorphan attenuates ethanol withdrawal syndrome in rats. Pharmacol Biochem Behav 62(3):537–541CrossRefGoogle Scholar
  30. 30.
    Liang X, Hu Q, Li B, McBride D, Bian H, Spagnoli P, Chen D, Tang J, Zhang JH (2014) Follistatin-like 1 attenuates apoptosis via disco-interacting protein 2 homolog A/Akt pathway after middle cerebral artery occlusion in rats. Stroke 45(10):3048–3054. CrossRefGoogle Scholar
  31. 31.
    Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning induces long-term potentiation in the hippocampus. Science 313(5790):1093–1097. CrossRefGoogle Scholar
  32. 32.
    Chandrasekar R (2013) Alcohol and NMDA receptor: current research and future direction. Front Mol Neurosci 6:14. CrossRefGoogle Scholar
  33. 33.
    Brady ML, Diaz MR, Iuso A, Everett JC, Valenzuela CF, Caldwell KK (2013) Moderate prenatal alcohol exposure reduces plasticity and alters NMDA receptor subunit composition in the dentate gyrus. J Neurosci 33(3):1062–1067. CrossRefGoogle Scholar
  34. 34.
    Roberto M, Bajo M, Crawford E, Madamba SG, Siggins GR (2006) Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amygdala. Neuropsychopharmacology 31(5):988–996. CrossRefGoogle Scholar
  35. 35.
    Miller CA, Sweatt JD (2007) Covalent modification of DNA regulates memory formation. Neuron 53(6):857–869. CrossRefGoogle Scholar
  36. 36.
    Law AJ, Weickert CS, Webster MJ, Herman MM, Kleinman JE, Harrison PJ (2003) Expression of NMDA receptor NR1, NR2A and NR2B subunit mRNAs during development of the human hippocampal formation. Eur J Neurosci 18(5):1197–1205CrossRefGoogle Scholar
  37. 37.
    Nakayama J, Rice JC, Strahl BD, Allis CD, Grewal SI (2001) Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292(5514):110–113. CrossRefGoogle Scholar
  38. 38.
    Yamada N, Nishida Y, Tsutsumida H, Hamada T, Goto M, Higashi M, Nomoto M, Yonezawa S (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 68(8):2708–2716. CrossRefGoogle Scholar
  39. 39.
    Park PH, Lim RW, Shukla SD (2005) Involvement of histone acetyltransferase (HAT) in ethanol-induced acetylation of histone H3 in hepatocytes: potential mechanism for gene expression. Am J Physiol Gastrointest Liver Physiol 289(6):G1124–G1136. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Duan Li
    • 1
    • 2
  • Yanqing Zhang
    • 1
  • Yanting Zhang
    • 1
  • Qi Wang
    • 1
  • Qin Miao
    • 1
  • Yahui Xu
    • 1
  • Jair C. Soares
    • 3
  • Xiangyang Zhang
    • 1
    • 3
    Email author
  • Ruiling Zhang
    • 1
    Email author
  1. 1.The Second Affiliated Hospital of Xinxiang Medical UniversityXinxiangPeople’s Republic of China
  2. 2.Department of Microbiology, School of Basic Medical SciencesXinxiang Medical UniversityXinxiangPeople’s Republic of China
  3. 3.Department of Psychiatry and Behavioral Sciences, McGovern Medical SchoolThe University of Texas Health Science Center at HoustonHoustonUSA

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