Molecular Neurobiology

, Volume 56, Issue 3, pp 1933–1945 | Cite as

Spatial Distribution and Expression of Ectonucleotidases in Rat Hippocampus After Removal of Ovaries and Estradiol Replacement

  • Ivana GrkovićEmail author
  • Nataša Mitrović
  • Milorad Dragić
  • Marija Adžić
  • Dunja Drakulić
  • Nadežda Nedeljković


Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as a fast excitatory transmitter, while adenosine sets a global inhibitory tone within hippocampal neuronal networks. ATP and adenosine are interconnected by ectonucleotidase enzymes, which convert ATP to adenosine. Existing data point to the converging roles of ovarian steroids and purinergic signaling in synapse formation and refinement and synapse activity in the hippocampus. Therefore, in the present study, we have used enzyme histochemistry and expression analysis to obtain data on spatial distribution and expression of ecto-enzymes NTPDase1, NTPDase2, and ecto-5′-nucleotidase (eN) after removal of ovaries (OVX) and estradiol replacement (E2) in female rat hippocampus. The results show that target ectonucleotidases are predominantly localized in synapse-rich hippocampal layers. The most represented NTPDase in the hippocampal tissue is NTPDase2, being at the same time the mostly affected ectonucleotidase by OVX and E2. Specifically, OVX decreases the expression of NTPDase2 and eN, whereas E2 restores their expression to control level. Impact of OVX and E2 on ectonucleotidase expression was also examined in purified synaptosome (SYN) and gliosome (GLIO) fractions. Data reveal that SYN expresses NTPDase1 and NTPDase2, both of which are reduced following OVX and restored with E2. GLIO exhibits NTPDase2-mediated ATP hydrolysis, which falls in OVX, and recovers by E2. These changes in the activity occur without parallel changes in NTPDase2-protein abundance. The same holds for eN. The lack of correlation between NTPDase2 and eN activities and their respective protein abundances suggest a non-genomic mode of E2 action, which is studied further in primary astrocyte culture. Since ovarian steroids shape hippocampal synaptic networks and regulate ectonucleotidase activities, it is possible that cognitive deficits seen after ovary removal may arise from the loss of E2 modulatory actions on ectonucleotidase expression in the hippocampus.


Ectonucleotidases Hippocampus Ovariectomy Estradiol Astrocytes 



The authors thank Dr. Terence Kirley from the University of Cincinnati, OH, USA, for a kind gift of rabbit anti-rat NTPDase2 and NTPDase3 antibodies used in this study.

Funding Information

The study was entirely supported by Ministry of Education, Science and Technological Development of the Republic of Serbia Nos. OI173044 and III41014.

Compliance with Ethical Standards

All experimental procedures involving rats were approved by the Ethical Committee for the Use of Laboratory Animals of Vinča Institute of Nuclear Sciences, Belgrade, Republic of Serbia (Application No. 02/11) and carried out according to the European Communities Council Directive (2010/63/EU).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2018_1217_Fig6_ESM.png (1.7 mb)
Supplementary Figure 1

Microglial cells in the stratum oriens, labeled with ATP enzyme histochemistry. (A) In control animals (Int), microglial cells exhibit typical small ovoid cell body with radially oriented ramified processes. (B) Removal of ovaries (OVX) induced visible changes the morphology of microglia, which became slightly enlarged and with a bipolar, lengthwise orientation of processes giving them the appearance of a less ramified cells. (C) Treatment with E2 restored highly ramified appearance similar to that found in control. Scale bar: 20 μm (PNG 1707 kb)

12035_2018_1217_MOESM1_ESM.tif (2.1 mb)
High Resolution Image (TIF 2160 kb)


  1. 1.
    Prange-Kiel J, Rune GM (2006) Direct and indirect effects of estrogen on rat hippocampus. Neuroscience 138(3):765–772. CrossRefGoogle Scholar
  2. 2.
    Lisofsky N, Martensson J, Eckert A, Lindenberger U, Gallinat J, Kuhn S (2015) Hippocampal volume and functional connectivity changes during the female menstrual cycle. NeuroImage 118:154–162. CrossRefGoogle Scholar
  3. 3.
    Woolley CS, McEwen BS (1993) Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J Comp Neurol 336(2):293–306. CrossRefGoogle Scholar
  4. 4.
    Brinton RD (2009) Estrogen-induced plasticity from cells to circuits: predictions for cognitive function. Trends Pharmacol Sci 30(4):212–222. CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Galea LA, Uban KA, Epp JR, Brummelte S, Barha CK, Wilson WL, Lieblich SE, Pawluski JL (2008) Endocrine regulation of cognition and neuroplasticity: our pursuit to unveil the complex interaction between hormones, the brain, and behaviour. Can J Exp Psychol 62(4):247–260. CrossRefGoogle Scholar
  6. 6.
    Fester L, Prange-Kiel J, Zhou L, Blittersdorf BV, Bohm J, Jarry H, Schumacher M, Rune GM (2012) Estrogen-regulated synaptogenesis in the hippocampus: sexual dimorphism in vivo but not in vitro. J Steroid Biochem Mol Biol 131(1–2):24–29. CrossRefGoogle Scholar
  7. 7.
    Frankfurt M, Luine V (2015) The evolving role of dendritic spines and memory: interaction(s) with estradiol. Horm Behav 74:28–36. CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Frick KM, Kim J, Tuscher JJ, Fortress AM (2015) Sex steroid hormones matter for learning and memory: estrogenic regulation of hippocampal function in male and female rodents. Learn Mem 22(9):472–493. CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Hara Y, Waters EM, McEwen BS, Morrison JH (2015) Estrogen effects on cognitive and synaptic health over the lifecourse. Physiol Rev 95(3):785–807. CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Sellers K, Raval P, Srivastava DP (2015) Molecular signature of rapid estrogen regulation of synaptic connectivity and cognition. Front Neuroendocrinol 36:72–89. CrossRefGoogle Scholar
  11. 11.
    Phan A, Suschkov S, Molinaro L, Reynolds K, Lymer JM, Bailey CD, Kow LM, MacLusky NJ et al (2015) Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements. Proc Natl Acad Sci U S A 112(52):16018–16023. CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Henderson VW (2008) Cognitive changes after menopause: influence of estrogen. Clin Obstet Gynecol 51(3):618–626. CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Krug R, Born J, Rasch B (2006) A 3-day estrogen treatment improves prefrontal cortex-dependent cognitive function in postmenopausal women. Psychoneuroendocrinology 31(8):965–975. CrossRefGoogle Scholar
  14. 14.
    Habib P, Beyer C (2015) Regulation of brain microglia by female gonadal steroids. J Steroid Biochem Mol Biol 146:3–14. CrossRefGoogle Scholar
  15. 15.
    Sarvari M, Kallo I, Hrabovszky E, Solymosi N, Liposits Z (2014) Ovariectomy and subsequent treatment with estrogen receptor agonists tune the innate immune system of the hippocampus in middle-aged female rats. PLoS One 9(2):e88540. CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Acaz-Fonseca E, Avila-Rodriguez M, Garcia-Segura LM, Barreto GE (2016) Regulation of astroglia by gonadal steroid hormones under physiological and pathological conditions. Prog Neurobiol 144:5–26. CrossRefGoogle Scholar
  17. 17.
    Hamilton RT, Rettberg JR, Mao Z, To J, Zhao L, Appt SE, Register TC, Kaplan JR et al (2011) Hippocampal responsiveness to 17beta-estradiol and equol after long-term ovariectomy: implication for a therapeutic window of opportunity. Brain Res 1379:11–22. CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Gillies GE, McArthur S (2010) Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines. Pharmacol Rev 62(2):155–198. CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Potier M, Georges F, Brayda-Bruno L, Ladepeche L, Lamothe V, Al Abed AS, Groc L, Marighetto A (2016) Temporal memory and its enhancement by estradiol requires surface dynamics of hippocampal CA1 N-methyl-D-aspartate receptors. Biol Psychiatry 79(9):735–745. CrossRefGoogle Scholar
  20. 20.
    Stanojlovic M, Gusevac I, Grkovic I, Mitrovic N, Zlatkovic J, Horvat A, Drakulic D (2016) Repeated estradiol treatment attenuates chronic cerebral hypoperfusion-induced neurodegeneration in rat hippocampus. Cell Mol Neurobiol 36(6):989–999. CrossRefGoogle Scholar
  21. 21.
    Sherwin BB (2012) Estrogen and cognitive functioning in women: lessons we have learned. Behav Neurosci 126(1):123–127. CrossRefGoogle Scholar
  22. 22.
    Schipper HM (2016) The impact of gonadal hormones on the expression of human neurological disorders. Neuroendocrinology 103(5):417–431. CrossRefGoogle Scholar
  23. 23.
    Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 87(2):659–797. CrossRefGoogle Scholar
  24. 24.
    Burnstock G (2016) An introduction to the roles of purinergic signalling in neurodegeneration, neuroprotection and neuroregeneration. Neuropharmacology 104:4–17. CrossRefGoogle Scholar
  25. 25.
    Koles L, Kato E, Hanuska A, Zadori ZS, Al-Khrasani M, Zelles T, Rubini P, Illes P (2016) Modulation of excitatory neurotransmission by neuronal/glial signalling molecules: interplay between purinergic and glutamatergic systems. Purinergic Signal 12(1):1–24. CrossRefGoogle Scholar
  26. 26.
    Sebastiao AM, Ribeiro JA (2015) Neuromodulation and metamodulation by adenosine: impact and subtleties upon synaptic plasticity regulation. Brain Res 1621:102–113. CrossRefGoogle Scholar
  27. 27.
    Zimmermann H, Zebisch M, Strater N (2012) Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 8(3):437–502. CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Robson SC, Sevigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signal 2(2):409–430. CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Langer D, Hammer K, Koszalka P, Schrader J, Robson S, Zimmermann H (2008) Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res 334(2):199–217. CrossRefGoogle Scholar
  30. 30.
    Grkovic I, Drakulic D, Martinovic J, Mitrovic N (2017) Role of ectonucleotidases in the synapse formation during brain development: physiological and pathological implications. Curr Neuropharmacol 15.
  31. 31.
    Bjelobaba I, Stojiljkovic M, Pekovic S, Dacic S, Lavrnja I, Stojkov D, Rakic L, Nedeljkovic N (2007) Immunohistological determination of ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and 5′-nucleotidase in rat hippocampus reveals overlapping distribution. Cell Mol Neurobiol 27(6):731–743. CrossRefGoogle Scholar
  32. 32.
    Wang TF, Guidotti G (1998) Widespread expression of ecto-apyrase (CD39) in the central nervous system. Brain Res 790(1–2):318–322CrossRefGoogle Scholar
  33. 33.
    Braun N, Sevigny J, Robson SC, Enjyoji K, Guckelberger O, Hammer K, Di Virgilio F, Zimmermann H (2000) Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. Eur J Neurosci 12(12):4357–4366Google Scholar
  34. 34.
    Brisevac D, Adzic M, Laketa D, Parabucki A, Milosevic M, Lavrnja I, Bjelobaba I, Sevigny J et al (2015) Extracellular ATP selectively upregulates ecto-nucleoside triphosphate diphosphohydrolase 2 and ecto-5'-nucleotidase by rat cortical astrocytes in vitro. J Mol Neurosci 57(3):452–462. CrossRefGoogle Scholar
  35. 35.
    Jakovljevic M, Lavrnja I, Bozic I, Savic D, Bjelobaba I, Pekovic S, Sevigny J, Nedeljkovic N et al (2017) Down-regulation of NTPDase2 and ADP-sensitive P2 purinoceptors correlate with severity of symptoms during experimental autoimmune encephalomyelitis. Front Cell Neurosci 11:333. CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Wink MR, Braganhol E, Tamajusuku AS, Lenz G, Zerbini LF, Libermann TA, Sevigny J, Battastini AM et al (2006) Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes. Neuroscience 138(2):421–432. CrossRefGoogle Scholar
  37. 37.
    Braun N, Sevigny J, Mishra SK, Robson SC, Barth SW, Gerstberger R, Hammer K, Zimmermann H (2003) Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain. Eur J Neurosci 17(7):1355–1364CrossRefGoogle Scholar
  38. 38.
    Shukla V, Zimmermann H, Wang L, Kettenmann H, Raab S, Hammer K, Sevigny J, Robson SC et al (2005) Functional expression of the ecto-ATPase NTPDase2 and of nucleotide receptors by neuronal progenitor cells in the adult murine hippocampus. J Neurosci Res 80(5):600–610. CrossRefGoogle Scholar
  39. 39.
    Belcher SM, Zsarnovszky A, Crawford PA, Hemani H, Spurling L, Kirley TL (2006) Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors. Neuroscience 137(4):1331–1346. CrossRefGoogle Scholar
  40. 40.
    Grkovic I, Bjelobaba I, Mitrovic N, Lavrnja I, Drakulic D, Martinovic J, Stanojlovic M, Horvat A et al (2016) Expression of ecto-nucleoside triphosphate diphosphohydrolase3 (NTPDase3) in the female rat brain during postnatal development. J Chem Neuroanat 77:10–18. CrossRefGoogle Scholar
  41. 41.
    Lavrnja I, Laketa D, Savic D, Bozic I, Bjelobaba I, Pekovic S, Nedeljkovic N (2015) Expression of a second ecto-5′-nucleotidase variant besides the usual protein in symptomatic phase of experimental autoimmune encephalomyelitis. J Mol Neurosci 55(4):898–911. CrossRefGoogle Scholar
  42. 42.
    Cunha RA (2001) Regulation of the ecto-nucleotidase pathway in rat hippocampal nerve terminals. Neurochem Res 26(8–9):979–991CrossRefGoogle Scholar
  43. 43.
    Kukulski F, Komoszynski M (2003) Purification and characterization of NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) from porcine brain cortex synaptosomes. Eur J Biochem 270(16):3447–3454CrossRefGoogle Scholar
  44. 44.
    Stanojevic I, Bjelobaba I, Nedeljkovic N, Drakulic D, Petrovic S, Stojiljkovic M, Horvat A (2011) Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes. Int J Dev Neurosci 29(4):397–403. CrossRefGoogle Scholar
  45. 45.
    Grkovic I, Bjelobaba I, Nedeljkovic N, Mitrovic N, Drakulic D, Stanojlovic M, Horvat A (2014) Developmental increase in ecto-5′-nucleotidase activity overlaps with appearance of two immunologically distinct enzyme isoforms in rat hippocampal synaptic plasma membranes. J Mol Neurosci 54(1):109–118. CrossRefGoogle Scholar
  46. 46.
    Mitrovic N, Zaric M, Drakulic D, Martinovic J, Sevigny J, Stanojlovic M, Nedeljkovic N, Grkovic I (2017) 17beta-estradiol-induced synaptic rearrangements are accompanied by altered ectonucleotidase activities in male rat hippocampal synaptosomes. J Mol Neurosci 61(3):412–422. CrossRefGoogle Scholar
  47. 47.
    Mitrovic N, Zaric M, Drakulic D, Martinovic J, Stanojlovic M, Sevigny J, Horvat A, Nedeljkovic N et al (2016) 17Beta-estradiol upregulates ecto-5′-nucleotidase (CD73) in hippocampal synaptosomes of female rats through action mediated by estrogen receptor-alpha and -beta. Neuroscience 324:286–296. CrossRefGoogle Scholar
  48. 48.
    Brisevac D, Bjelobaba I, Bajic A, Clarner T, Stojiljkovic M, Beyer C, Andjus P, Kipp M et al (2012) Regulation of ecto-5′-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors. Neurochem Int 61(5):681–688. CrossRefGoogle Scholar
  49. 49.
    Da Silva RS, Richetti SK, Tonial EM, Bogo MR, Bonan CD (2012) Profile of nucleotide catabolism and ectonucleotidase expression from the hippocampi of neonatal rats after caffeine exposure. Neurochem Res 37(1):23–30. CrossRefGoogle Scholar
  50. 50.
    Mitrovic N, Gusevac I, Drakulic D, Stanojlovic M, Zlatkovic J, Sevigny J, Horvat A, Nedeljkovic N et al (2016) Regional and sex-related differences in modulating effects of female sex steroids on ecto-5′-nucleotidase expression in the rat cerebral cortex and hippocampus. Gen Comp Endocrinol 235:100–107. CrossRefGoogle Scholar
  51. 51.
    Milanese M, Bonifacino T, Zappettini S, Usai C, Tacchetti C, Nobile M, Bonanno G (2009) Glutamate release from astrocytic gliosomes under physiological and pathological conditions. Int Rev Neurobiol 85:295–318. CrossRefGoogle Scholar
  52. 52.
    Bari M, Bonifacino T, Milanese M, Spagnuolo P, Zappettini S, Battista N, Giribaldi F, Usai C et al (2011) The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release. Cell Mol Life Sci 68(5):833–845. CrossRefGoogle Scholar
  53. 53.
    Corera AT, Doucet G, Fon EA (2009) Long-term potentiation in isolated dendritic spines. PLoS One 4(6):e6021. CrossRefPubMedCentralPubMedGoogle Scholar
  54. 54.
    Heine P, Braun N, Heilbronn A, Zimmermann H (1999) Functional characterization of rat ecto-ATPase and ecto-ATP diphosphohydrolase after heterologous expression in CHO cells. Eur J Biochem 262(1):102–107CrossRefGoogle Scholar
  55. 55.
    Wink MR, Braganhol E, Tamajusuku AS, Casali EA, Karl J, Barreto-Chaves ML, Sarkis JJ, Battastini AM (2003) Extracellular adenine nucleotides metabolism in astrocyte cultures from different brain regions. Neurochem Int 43(7):621–628CrossRefGoogle Scholar
  56. 56.
    Matyash M, Zabiegalov O, Wendt S, Matyash V, Kettenmann H (2017) The adenosine generating enzymes CD39/CD73 control microglial processes ramification in the mouse brain. PLoS One 12(4):e0175012. CrossRefPubMedCentralPubMedGoogle Scholar
  57. 57.
    Vlajkovic SM, Housley GD, Greenwood D, Thorne PR (1999) Evidence for alternative splicing of ecto-ATPase associated with termination of purinergic transmission. Brain Res Mol Brain Res 73(1–2):85–92CrossRefGoogle Scholar
  58. 58.
    Vlajkovic SM, Wang CJ, Soeller C, Zimmermann H, Thorne PR, Housley GD (2007) Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells. Int J Biochem Cell Biol 39(4):810–817. CrossRefGoogle Scholar
  59. 59.
    Wang CJ, Vlajkovic SM, Housley GD, Braun N, Zimmermann H, Robson SC, Sevigny J, Soeller C et al (2005) C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation. Biochem J 385(Pt 3):729–736. CrossRefPubMedCentralPubMedGoogle Scholar
  60. 60.
    Hansen KR, Resta R, Webb CF, Thompson LF (1995) Isolation and characterization of the promoter of the human 5′-nucleotidase (CD73)-encoding gene. Gene 167(1–2):307–312CrossRefGoogle Scholar
  61. 61.
    Spychala J, Lazarowski E, Ostapkowicz A, Ayscue LH, Jin A, Mitchell BS (2004) Role of estrogen receptor in the regulation of ecto-5′-nucleotidase and adenosine in breast cancer. Clin Cancer Res 10(2):708–717CrossRefGoogle Scholar
  62. 62.
    Zelinski DP, Zantek ND, Walker-Daniels J, Peters MA, Taparowsky EJ, Kinch MS (2002) Estrogen and Myc negatively regulate expression of the EphA2 tyrosine kinase. J Cell Biochem 85(4):714–720. CrossRefGoogle Scholar
  63. 63.
    Vivar OI, Zhao X, Saunier EF, Griffin C, Mayba OS, Tagliaferri M, Cohen I, Speed TP et al (2010) Estrogen receptor beta binds to and regulates three distinct classes of target genes. J Biol Chem 285(29):22059–22066. CrossRefPubMedCentralPubMedGoogle Scholar
  64. 64.
    Spychala J, Zimmermann AG, Mitchell BS (1999) Tissue-specific regulation of the ecto-5′-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region. J Biol Chem 274(32):22705–22712CrossRefGoogle Scholar
  65. 65.
    Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, Brodsky AS, Keeton EK et al (2006) Genome-wide analysis of estrogen receptor binding sites. Nat Genet 38(11):1289–1297. CrossRefGoogle Scholar
  66. 66.
    Spychala J, Mitchell BS, Barankiewicz J (1997) Adenosine metabolism during phorbol myristate acetate-mediated induction of HL-60 cell differentiation: changes in expression pattern of adenosine kinase, adenosine deaminase, and 5′-nucleotidase. J Immunol 158(10):4947–4952Google Scholar
  67. 67.
    Milner TA, Ayoola K, Drake CT, Herrick SP, Tabori NE, McEwen BS, Warrier S, Alves SE (2005) Ultrastructural localization of estrogen receptor beta immunoreactivity in the rat hippocampal formation. J Comp Neurol 491(2):81–95. CrossRefGoogle Scholar
  68. 68.
    Bilbao PS, Katz S, Boland R (2012) Interaction of purinergic receptors with GPCRs, ion channels, tyrosine kinase and steroid hormone receptors orchestrates cell function. Purinergic Signal 8(1):91–103. CrossRefGoogle Scholar
  69. 69.
    Campanac E, Gasselin C, Baude A, Rama S, Ankri N, Debanne D (2013) Enhanced intrinsic excitability in basket cells maintains excitatory-inhibitory balance in hippocampal circuits. Neuron 77(4):712–722. CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Molecular Biology and Endocrinology, Vinča Institute of Nuclear SciencesUniversity of BelgradeBelgradeSerbia
  2. 2.Department for General Physiology and Biophysics, Faculty of BiologyUniversity of BelgradeBelgradeSerbia

Personalised recommendations