Molecular Neurobiology

, Volume 55, Issue 11, pp 8738–8753 | Cite as

Hypoxia-Induced Neuroinflammation and Learning–Memory Impairments in Adult Zebrafish Are Suppressed by Glucosamine

  • Yunkyoung Lee
  • Sujeong Lee
  • Ji-Won Park
  • Ji-Sun Hwang
  • Sang-Min Kim
  • In Kyoon Lyoo
  • Chang-Joong Lee
  • Inn-Oc Han


This study investigated changes in neuroinflammation and cognitive function in adult zebrafish exposed to acute hypoxia and protective effects of glucosamine (GlcN) against hypoxia-induced brain damage. The survival rate of zebrafish following exposure to hypoxia was improved by GlcN pretreatment. Moreover, hypoxia-induced upregulation of neuroglobin, NOS2α, glial fibrillary acidic protein, and S100β in zebrafish was suppressed by GlcN. Hypoxia stimulated cell proliferation in the telencephalic ventral domain and in cerebellum subregions. GlcN decreased the number of bromodeoxyuridine (BrdU)-positive cells in the telencephalon region, but not in cerebellum regions. Transient motor neuron defects, assessed by measuring the locomotor and exploratory activity of zebrafish exposed to hypoxia recovered quickly. GlcN did not affect hypoxia-induced motor activity changes. In passive avoidance tests, hypoxia impaired learning and memory ability, deficits that were rescued by GlcN. A learning stimulus increased the nuclear translocation of phosphorylated cAMP response element binding protein (p-CREB), an effect that was greatly inhibited by hypoxia. GlcN restored nuclear p-CREB after a learning trial in hypoxia-exposed zebrafish. The neurotransmitters, γ-aminobutyric acid and glutamate, were increased after hypoxia in the zebrafish brain, and GlcN further increased their levels. In contrast, acetylcholine levels were reduced by hypoxia and restored by GlcN. Acetylcholinesterase inhibitor physostigmine partially reversed the impaired learning and memory of hypoxic zebrafish. This study represents the first examination of the molecular mechanisms underlying hypoxia-induced memory and learning defects in a zebrafish model. Our results further suggest that GlcN-associated hexosamine metabolic pathway could be an important therapeutic target for hypoxic brain damage.


Hypoxia Glucosamine Neuroinflammation CREB Zebrafish 


Author Contributions

IO Han designed the study and supervised the project; Y Lee and S Lee wrote the manuscript and analyzed the data; JW Park, SM Kim and JS Hwang performed experiments and the imaging studies; CJ Lee and IK Lyoo contributed to designing experimental procedures. All authors reviewed the manuscript.

Funding Information

This work was supported by the National Research Foundation (NRF) of Korea grants, NRF-2017R1A2B2007199, NRF-2017R1D1A1B03031431, and the Brain Research Program 2015M3C7A1028373 and WCSL (World Class Smart Lab) research grant of Inha University.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12035_2018_1017_Fig10_ESM.gif (46 kb)
Supplemental Figure 1

Change of O-GlcNAc level in the brain of adult zebrafish in response to hypoxia with or without GlcN pretreatment. Adult zebrafish were pretreated with 1 g/L GlcN for 12 h and then exposed to hypoxic conditions for 8 min. After a 6- or 24-h recovery period, total protein extraction from brain were prepared and O-GlcNAc protein level was determined by Western blotting along with ⍺-Tubulin as a loading control. Shown blots are representatives of three independent experiments. All values are means ± SEM. *P < 0.05 versus C; #P < 0.05 versus H. A.U., arbitrary units; C, control (normoxia); H, hypoxia; G, GlcN; HG, hypoxia + GlcN. (GIF 45 kb)

12035_2018_1017_MOESM1_ESM.tif (7.9 mb)
High resolution image (TIFF 8128 kb)


  1. 1.
    Chiu GS, Chatterjee D, Darmody PT, Walsh JP, Meling DD, Johnson RW, Freund GG (2012) Hypoxia/reoxygenation impairs memory formation via adenosine-dependent activation of caspase 1. J Neurosci 32:13945–13955. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Beatty WW, Salmon DP, Bernstein N, Martone M, Lyon L, Butters N (1987) Procedural learning in a patient with amnesia due to hypoxia. Brain Cogn 6:386–402CrossRefGoogle Scholar
  3. 3.
    Braga MM, Rico EP, Cordova SD, Pinto CB, Blaser RE, Dias RD, Rosemberg DB, Oliveira DL et al (2013) Evaluation of spontaneous recovery of behavioral and brain injury profiles in zebrafish after hypoxia. Behav Brain Res 253:145–151. CrossRefPubMedGoogle Scholar
  4. 4.
    Braga MM, Silva ES, Moraes TB, Schirmbeck GH, Rico EP, Pinto CB, Rosemberg DB, Dutra-Filho CS et al (2016) Brain zinc chelation by diethyldithiocarbamate increased the behavioral and mitochondrial damages in zebrafish subjected to hypoxia. Sci Rep 6:20279. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Shukitt-Hale B, Stillman MJ, Lieberman HR (1996) Tyrosine administration prevents hypoxia-induced decrements in learning and memory. Physiol Behav 59:867–871CrossRefGoogle Scholar
  6. 6.
    Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M, Mori K, Ikeda T et al (2008) Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat Neurosci 11:1153–1161. CrossRefPubMedGoogle Scholar
  7. 7.
    Kaslin J, Ganz J, Brand M (2008) Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain. Philos Trans R Soc Lond Ser B Biol Sci 363:101–122. CrossRefGoogle Scholar
  8. 8.
    Altman J (1969) Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 137:433–457. CrossRefPubMedGoogle Scholar
  9. 9.
    Zupanc GK, Hinsch K, Gage FH (2005) Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain. J Comp Neurol 488:290–319. CrossRefPubMedGoogle Scholar
  10. 10.
    Grandel H, Kaslin J, Ganz J, Wenzel I, Brand M (2006) Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. Dev Biol 295:263–277. CrossRefPubMedGoogle Scholar
  11. 11.
    Azuma K, Osaki T, Wakuda T, Tsuka T, Imagawa T, Okamoto Y, Minami S (2012) Suppressive effects of N-acetyl-D-glucosamine on rheumatoid arthritis mouse models. Inflammation 35:1462–1465. CrossRefPubMedGoogle Scholar
  12. 12.
    Ma L, Rudert WA, Harnaha J, Wright M, Machen J, Lakomy R, Qian S, Lu L et al (2002) Immunosuppressive effects of glucosamine. J Biol Chem 277:39343–39349. CrossRefPubMedGoogle Scholar
  13. 13.
    Champattanachai V, Marchase RB, Chatham JC (2007) Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc. Am J Physiol Cell Physiol 292:C178–C187. CrossRefPubMedGoogle Scholar
  14. 14.
    Liu J, Pang Y, Chang T, Bounelis P, Chatham JC, Marchase RB (2006) Increased hexosamine biosynthesis and protein O-GlcNAc levels associated with myocardial protection against calcium paradox and ischemia. J Mol Cell Cardiol 40:303–312. CrossRefPubMedGoogle Scholar
  15. 15.
    Zupanets IA, Pliushch SI, Drogovoz SM, Zagrebely D (1992) Antihypoxic effect of glucosamine in acute hypoxic states in mice. Fiziol Zh 38:88–90PubMedGoogle Scholar
  16. 16.
    Hwang SY, Shin JH, Hwang JS, Kim SY, Shin JA, Oh ES, Oh S, Kim JB et al (2010) Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury. Glia 58:1881–1892. CrossRefPubMedGoogle Scholar
  17. 17.
    Algra SO, Groeneveld KM, Schadenberg AW, Haas F, Evens FC, Meerding J, Koenderman L, Jansen NJ et al (2013) Cerebral ischemia initiates an immediate innate immune response in neonates during cardiac surgery. J Neuroinflammation 10:24. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Petito CK, Morgello S, Felix JC, Lesser ML (1990) The two patterns of reactive astrocytosis in postischemic rat brain. J Cereb Blood Flow Metab 10:850–859. CrossRefPubMedGoogle Scholar
  19. 19.
    Morris RG, Anderson E, Lynch GS, Baudry M (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774–776. CrossRefPubMedGoogle Scholar
  20. 20.
    Reymann KG, Frey JU (2007) The late maintenance of hippocampal LTP: requirements, phases, ‘synaptic tagging’, ‘late-associativity’ and implications. Neuropharmacology 52:24–40. CrossRefPubMedGoogle Scholar
  21. 21.
    Kida S, Serita T (2014) Functional roles of CREB as a positive regulator in the formation and enhancement of memory. Brain Res Bull 105:17–24. CrossRefPubMedGoogle Scholar
  22. 22.
    Carlezon WA Jr, Duman RS, Nestler EJ (2005) The many faces of CREB. Trends Neurosci 28:436–445. CrossRefPubMedGoogle Scholar
  23. 23.
    Shaywitz AJ, Greenberg ME (1999) CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem 68:821–861. CrossRefPubMedGoogle Scholar
  24. 24.
    Josselyn SA, Nguyen PV (2005) CREB, synapses and memory disorders: past progress and future challenges. Curr Drug Targets CNS Neurol Disord 4:481–497CrossRefGoogle Scholar
  25. 25.
    Trifilieff P, Herry C, Vanhoutte P, Caboche J, Desmedt A, Riedel G, Mons N, Micheau J (2006) Foreground contextual fear memory consolidation requires two independent phases of hippocampal ERK/CREB activation. Learn Mem 13:349–358. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mizuno M, Yamada K, Maekawa N, Saito K, Seishima M, Nabeshima T (2002) CREB phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behav Brain Res 133:135–141CrossRefGoogle Scholar
  27. 27.
    Cammarota M, Bevilaqua LR, Ardenghi P, Paratcha G, Levi de Stein M, Izquierdo I, Medina JH (2000) Learning-associated activation of nuclear MAPK, CREB and Elk-1, along with Fos production, in the rat hippocampus after a one-trial avoidance learning: abolition by NMDA receptor blockade. Brain Res Mol Brain Res 76:36–46CrossRefGoogle Scholar
  28. 28.
    Tropea TF, Kosofsky BE, Rajadhyaksha AM (2008) Enhanced CREB and DARPP-32 phosphorylation in the nucleus accumbens and CREB, ERK, and GluR1 phosphorylation in the dorsal hippocampus is associated with cocaine-conditioned place preference behavior. J Neurochem 106:1780–1790. CrossRefPubMedGoogle Scholar
  29. 29.
    Barros TP, Alderton WK, Reynolds HM, Roach AG, Berghmans S (2008) Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery. Br J Pharmacol 154:1400–1413. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Senger MR, Rosemberg DB, Rico EP, de Bem Arizi M, Dias RD, Bogo MR, Bonan CD (2006) In vitro effect of zinc and cadmium on acetylcholinesterase and ectonucleotidase activities in zebrafish (Danio rerio) brain. Toxicol in Vitro 20:954–958. CrossRefPubMedGoogle Scholar
  31. 31.
    Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, Elegante MF, Elkhayat SI, Bartels BK et al (2009) Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Res 205:38–44. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Yu X, Li YV (2011) Zebrafish as an alternative model for hypoxic-ischemic brain damage. Int J Physiol Pathophysiol Pharmacol 3:88–96PubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhang L, Li LH, Qu Y, Mu DZ (2008) Neuroglobin and hypoxic-ischemic brain bamage. Zhongguo Dang Dai Er Ke Za Zhi 10:265–268PubMedGoogle Scholar
  34. 34.
    Sun Y, Jin K, Mao XO, Zhu Y, Greenberg DA (2001) Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury. Proc Natl Acad Sci U S A 98:15306–15311. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci 20:570–577CrossRefGoogle Scholar
  36. 36.
    Iadecola C, Zhang F, Xu S, Casey R, Ross ME (1995) Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. J Cereb Blood Flow Metab 15:378–384. CrossRefPubMedGoogle Scholar
  37. 37.
    Kim YH, Lee Y, Kim D, Jung MW, Lee CJ (2010) Scopolamine-induced learning impairment reversed by physostigmine in zebrafish. Neurosci Res 67:156–161. CrossRefPubMedGoogle Scholar
  38. 38.
    Johnston MV (1983) Neurotransmitter alterations in a model of perinatal hypoxic-ischemic brain injury. Ann Neurol 13:511–518. CrossRefPubMedGoogle Scholar
  39. 39.
    Zupanc GK (2001) Adult neurogenesis and neuronal regeneration in the central nervous system of teleost fish. Brain Behav Evol 58:250–275CrossRefGoogle Scholar
  40. 40.
    Ferriero DM (2004) Neonatal brain injury. N Engl J Med 351:1985–1995. CrossRefPubMedGoogle Scholar
  41. 41.
    Farina C, Aloisi F, Meinl E (2007) Astrocytes are active players in cerebral innate immunity. Trends Immunol 28:138–145. CrossRefPubMedGoogle Scholar
  42. 42.
    Sawaguchi T, Patricia F, Kadhim H, Groswasser J, Sottiaux M, Nishida H, Kahn A (2003) Clinicopathological correlation between brainstem gliosis using GFAP as a marker and sleep apnea in the sudden infant death syndrome. Early Hum Dev 75(Suppl):S3–S11CrossRefGoogle Scholar
  43. 43.
    Privat A (2003) Astrocytes as support for axonal regeneration in the central nervous system of mammals. Glia 43:91–93. CrossRefPubMedGoogle Scholar
  44. 44.
    Stoll G, Jander S, Schroeter M (1998) Inflammation and glial responses in ischemic brain lesions. Prog Neurobiol 56:149–171CrossRefGoogle Scholar
  45. 45.
    Champattanachai V, Marchase RB, Chatham JC (2008) Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2. Am J Physiol Cell Physiol 294:C1509–C1520. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Newman MF, Kirchner JL, Phillips-Bute B, Gaver V, Grocott H, Jones RH, Mark DB, Reves JG et al (2001) Longitudinal assessment of neurocognitive function after coronary-artery bypass surgery. N Engl J Med 344:395–402. CrossRefPubMedGoogle Scholar
  47. 47.
    Myhrer T (2003) Neurotransmitter systems involved in learning and memory in the rat: a meta-analysis based on studies of four behavioral tasks. Brain Res Brain Res Rev 41:268–287CrossRefGoogle Scholar
  48. 48.
    Picciotto MR, Higley MJ, Mineur YS (2012) Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior. Neuron 76:116–129. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Campbell N, Boustani M, Limbil T, Ott C, Fox C, Maidment I, Schubert CC, Munger S et al (2009) The cognitive impact of anticholinergics: a clinical review. Clin Interv Aging 4:225–233PubMedPubMedCentralGoogle Scholar
  50. 50.
    Hagberg H, Lehmann A, Sandberg M, Nystrom B, Jacobson I, Hamberger A (1985) Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 5:413–419. CrossRefPubMedGoogle Scholar
  51. 51.
    Gozal D, Daniel JM, Dohanich GP (2001) Behavioral and anatomical correlates of chronic episodic hypoxia during sleep in the rat. J Neurosci 21:2442–2450CrossRefGoogle Scholar
  52. 52.
    Aviles-Reyes RX, Angelo MF, Villarreal A, Rios H, Lazarowski A, Ramos AJ (2010) Intermittent hypoxia during sleep induces reactive gliosis and limited neuronal death in rats: implications for sleep apnea. J Neurochem 112:854–869. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Physiology and Biophysics, College of MedicineInha UniversityIncheonSouth Korea
  2. 2.Department of Biological SciencesInha UniversityIncheonSouth Korea
  3. 3.Department of Brain and Cognitive Sciences, Ewha Brain Institute, College of PharmacyEwha Womans UniversitySeoulSouth Korea

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