Journal Mobile Options
Table of Contents
Vol. 80, No. 5, 2004
Issue release date: 2004
Neuroendocrinology 2004;80:308–323

Estrogen and Ovariectomy Regulate mRNA and Protein of Glutamic Acid Decarboxylases and Cation-Chloride Cotransporters in the Adult Rat Hippocampus

Nakamura N.H. · Rosell D.R. · Akama K.T. · McEwen B.S.
Laboratory of Neuroendocrinology, Rockefeller University, New York, N.Y., USA

Individual Users: Register with Karger Login Information

Please create your User ID & Password

Contact Information

I have read the Karger Terms and Conditions and agree.

To view the fulltext, please log in

To view the pdf, please log in


17β-Estradiol spatiotemporally regulates the γ-aminobutyric acid (GABAergic) tone in the adult hippocampus. However, the complex estrogenic effect on the GABAergic system is still unclear. In adult central nervous system (CNS) neurons, GABA can induce both inhibitory and excitatory actions, which are predominantly controlled by the cation-chloride cotransporters NKCC1 and KCC2. We therefore studied the estrogenic regulation of two glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, as well as NKCC1 and KCC2 in the adult female rat hippocampus by immunohistochemistry and in situ hybridization. First, we focused on the duration after ovariectomy (OVX) and its effects on GAD65 protein levels. The basal number of GAD65-immunoreactive cells decreased after long-term (10 days) OVX compared to short-term (3 days) OVX. We found that, only after long-term OVX but not after short-term OVX, estradiol increased the number of GAD65-immunoreactive cells in the CA1 pyramidal cell layer. Furthermore, estradiol did not alter the GAD65-immunoreactive cell population in any other CA1 subregion. Second, we therefore focused on long-term OVX and the estrogenic regulation of GAD and cation-chloride cotransporter mRNA levels. In the pyramidal cell layer, estradiol affected GAD65, GAD67 and NKCC1 mRNA levels, but not KCC2 mRNA levels. Both GAD65 and NKCC1 mRNA levels increased within 24 h after estradiol treatment, followed by a subsequent increase in GAD67 mRNA levels. These findings suggest that basal levels of estrogen might contribute to a balance between the excitatory and inhibitory synaptic transmission onto CA1 pyramidal cells by regulating perisomatic GAD and NKCC1 expression in the adult hippocampus.

Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.


  1. Woolley CS: Estrogen-mediated structural and functional synaptic plasticity in the female rat hippocampus. Horm Behav 1998;34:140–148.
  2. Segal M, Murphy D: Estradiol induces formation of dendritic spines in hippocampal neurons: Functional correlates. Horm Behav 2001;40:156–159.
  3. Terasawa E, Timiras PS: Electrical activity during the estrous cycle of the rat: Cyclic changes in limbic structures. Endocrinology 1968;83:207–216.
  4. Sandstrom NJ, Williams CL: Memory retention is modulated by acute estradiol and progesterone replacement. Behav Neurosci 2001;115:384–393.
  5. Milner TA, McEwen BS, Hayashi S, Li CJ, Reagan LP, Alves SE: Ultrastructural evidence that hippocampal alpha estrogen receptors are located at extranuclear sites. J Comp Neurol 2001;429:355–371.
  6. McEwen BS, Akama K, Alves S, Brake WG, Bulloch K, Lee S, Li C, Yuen G, Milner TA: Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation. Proc Natl Acad Sci USA 2001;98:7093–7100.
  7. McEwen BS: Invited review: Estrogens effects on the brain: Multiple sites and molecular mechanisms. J Appl Physiol 2001;91:2785–2801.
  8. Freund TF, Buzsaki G: Interneurons of the hippocampus. Hippocampus 1996;6:347–470.
  9. Weiland NG, Orikasa C, Hayashi S, McEwen BS: Distribution and hormone regulation of estrogen receptor immunoreactive cells in the hippocampus of male and female rats. J Comp Neurol 1997;388:603–612.
  10. Loy R, Gerlach JL, McEwen BS: Autoradiographic localization of estradiol-binding neurons in the rat hippocampal formation and entorhinal cortex. Brain Res 1988;467:245–251.
  11. Shughrue PJ, Merchenthaler I: Evidence for novel estrogen binding sites in the rat hippocampus. Neuroscience 2000;99:605–612.
  12. Shughrue PJ, Merchenthaler I: Distribution of estrogen receptor beta immunoreactivity in the rat central nervous system. J Comp Neurol 2001;436:64–81.
  13. Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, Pfaff DW, Ogawa S, Rohrer SP, Schaeffer JM, McEwen BS, Alves SE: Immunolocalization of estrogen receptor beta in the mouse brain: Comparison with estrogen receptor alpha. Endocrinology 2003;144:2055–2067.
  14. Murphy DD, Cole NB, Greenberger V, Segal M: Estradiol increases dendritic spine density by reducing GABA neurotransmission in hippocampal neurons. J Neurosci 1998;18:2550–2559.
  15. Rudick CN, Woolley CS: Estrogen regulates functional inhibition of hippocampal CA1 pyramidal cells in the adult female rat. J Neurosci 2001;21:6532–6543.
  16. Paulsen O, Moser EI: A model of hippocampal memory encoding and retrieval: GABAergic control of synaptic plasticity. Trends Neurosci 1998;21:273–278.
  17. Weiland NG: Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus. Endocrinology 1992;131:2697–2702.
  18. Schumacher M, Coirini H, McEwen BS: Regulation of high-affinity GABAA receptors in the dorsal hippocampus by estradiol and progesterone. Brain Res 1989;487:178–83.
  19. McCarthy MM, Coirini H, Johnson AE, Pfaff DW, Schwartz-Giblin S. McEwen BS: Steroid regulation and sex differences in [3H]muscimol binding in hippocampus, hypothalamus and midbrain in rats. J Neuroendocrinol 1992;4:393–399.
  20. Rudick CN, Woolley CS: Selective estrogen receptor modulators regulate phasic activation of hippocampal CA1 pyramidal cells by estrogen. Endocrinology 2003;144:179–187.
  21. Ben-Ari Y: Excitatory actions of GABA during development: The nature of the nurture. Nat Rev Neurosci 2002;3:728–739.
  22. Owens DF, Kriegstein AR: Is there more to GABA than synaptic inhibition? Nat Rev Neurosci 2002;3:715–727.
  23. Vardi N, Zhang LL, Payne JA, Sterling P: Evidence that different cation chloride cotransporters in retinal neurons allow opposite responses to GABA. J Neurosci 2000;20:7657–7663.
  24. Grover LM, Lambert NA, Schwartzkroin PA, Teyler TJ: Role of HCO3 ions in depolarizing GABAA receptor-mediated responses in pyramidal cells of rat hippocampus. J Neurophysiol 1993;69:1541–1555.
  25. Staley KJ, Soldo BL, Proctor WR: Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors. Science 1995;269:977–981.
  26. Kaila K, Lamsa K, Smirnov S, Taira T, Voipio J: Long-lasting GABA-mediated depolarization evoked by high-frequency stimulation in pyramidal neurons of rat hippocampal slice is attributable to a network-driven, bicarbonate-dependent K+ transient. J Neurosci 1997;17:7662–7672.
  27. Taira T, Lamsa K, Kaila K: Posttetanic excitation mediated by GABAA receptors in rat CA1 pyramidal neurons. J Neurophysiol 1997;77:2213–2218.
  28. Isomura Y, Sugimoto M, Fujiwara-Tsukamoto Y, Yamamoto-Muraki S, Yamada J, Fukuda A: Synaptically activated Cl accumulation responsible for depolarizing GABAergic responses in mature hippocampal neurons. J Neurophysiol 2003;90:2752–2756.
  29. Gulledge AT, Stuart GJ: Excitatory actions of GABA in the cortex. Neuron 2003;37:299–309.
  30. Chavas J, Marty A: Coexistence of excitatory and inhibitory GABA synapses in the cerebellar interneuron network. J Neurosci 2003;23:2019–2031.
  31. Stein V, Nicoll RA: GABA generates excitement. Neuron 2003;37:375–378.
  32. Payne JA, Rivera C, Voipio J, Kaila K: Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci 2003;26:199–206.
  33. Nakamura N, Fujita H, Kawata M: Effects of gonadectomy on immunoreactivity for choline acetyltransferase in the cortex, hippocampus, and basal forebrain of adult male rats. Neuroscience 2002;109:473–485.
  34. Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, Houser CR: Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 1994;14:1834–1855.
  35. Sloviter RS, Dichter MA, Rachinsky TL, Dean E, Goodman JH, Sollas AL, Martin DL: Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus. J Comp Neurol 1996;373:593–618.
  36. Moffatt CA, Rissman EF, Shupnik MA, Blaustein JD: Induction of progestin receptors by estradiol in the forebrain of estrogen receptor-alpha gene-disrupted mice. J Neurosci 1998;18:9556–9563.
  37. Schreihofer DA, Resnick EM, Soh AY, Shupnik MA: Transcriptional regulation by a naturally occurring truncated rat estrogen receptor (ER), truncated ER product-1 (TERP-1). Mol Endocrinol 1999;13:320–329.
  38. Solum DT, Handa RJ: Localization of estrogen receptor alpha (ER alpha) in pyramidal neurons of the developing rat hippocampus. Dev Brain Res 2001;128:165–175.
  39. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates, ed 4. San Diego, Academic Press, 1998.
  40. Guillery RW, Herrup K: Quantification without pontification: Choosing a method for counting objects in sectioned tissues. J Comp Neurol 1997;386:2–7.
  41. Mufson EJ, Cai WJ, Jaffar S, Chen E, Stebbins G, Sendera T, Kordower JH: Estrogen receptor immunoreactivity within subregions of the rat forebrain: Neuronal distribution and association with perikarya containing choline acetyltransferase. Brain Res 1999;849:253–274.
  42. Woolley CS, Wenzel HJ, Schwartzkroin PA: Estradiol increases the frequency of multiple synapse boutons in the hippocampal CA1 region of the adult female rat. J Comp Neurol 1996;373:108–117.
  43. Yankova M, Hart SA, Woolley CS: Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: A serial electron-microscopic study. Proc Natl Acad Sci USA 2001;98:3525–3530
  44. Gould E, Woolley CS, Frankfurt M, McEwen BS: Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. J Neurosci 1990;10:1286–1291.
  45. Woolley CS, McEwen BS: Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J Comp Neurol 1993;336:293–306.
  46. Woolley CS, Weiland NG, McEwen BS, Schwartzkroin PA: Estradiol increases the sensitivity of hippocampal CA1 pyramidal cells to NMDA receptor-mediated synaptic input: Correlation with dendritic spine density. J Neurosci 1997;17:1848–1859.
  47. Daniel JM, Dohanich GP: Acetylcholine mediates the estrogen-induced increase in NMDA receptor binding in CA1 of the hippocampus and the associated improvement in working memory. J Neurosci 2001;21:6949–6956.
  48. Rudick CN, Woolley CS: Estradiol induces a phasic Fos response in the hippocampal CA1 and CA3 regions of adult female rats. Hippocampus 2000;10:274–283.
  49. Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ: Two genes encode distinct glutamate decarboxylases. Neuron 1991;7:91–100.
  50. Clayton GH, Owens GC, Wolff JS, Smith RL: Ontogeny of cation-Cl cotransporter expression in rat neocortex. Brain Res Dev Brain Res 1998;109:281–292.
  51. Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K: The K+/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 1999;397:251–255.
  52. Reagan LP, McKittrick CR, McEwen BS: Corticosterone and phenytoin reduce neuronal nitric oxide synthase messenger RNA expression in rat hippocampus. Neuroscience 1999;91:211–219.
  53. Rosell DR, Nacher J, Akama KT, McEwen BS: Spatiotemporal distribution of gp130 cytokines and their receptors after status epilepticus: Comparison with neuronal degeneration and microglial activation. Neuroscience 2003;122:329–348.
  54. Du F, Eid T, Lothman EW, Kohler C, Schwarcz R: Preferential neuronal loss in layer III of the medial entorhinal cortex in rat models of temporal lobe epilepsy. J Neurosci 1995;15:6301–6313.
  55. Lucas LR, Angulo JA, Le Moal M, McEwen BS, Piazza P: Neurochemical characterization of individual vulnerability to addictive drugs in rats. Eur J Neurosci 1998;10:3153–3163.
  56. Feldblum S, Erlander MG, Tobin AJ: Different distributions of GAD65 and GAD67 mRNAs suggest that the two glutamate decarboxylases play distinctive functional roles. J Neurosci Res 1993;34:689–706.
  57. Houser CR, Esclapez M: Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation. Hippocampus 1994;4:530–545.
  58. Stone DJ, Walsh J, Benes FM: Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus: A double in situ hybridization study. Mol Brain Res 1999;71:201–209.
  59. Kanaka C, Ohno K, Okabe A, Kuriyama K, Itoh T, Fukuda A, Sato K: The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system. Neuroscience 2001;104:933–946.
  60. Wang C, Shimizu-Okabe C, Watanabe K, Okabe A, Matsuzaki H, Ogawa T, Mori N, Fukuda A, Sato K: Developmental changes in KCC1, KCC2, and NKCC1 mRNA expressions in the rat brain. Brain Res Dev Brain Res 2002;139:59–66.
  61. Soghomonian JJ, Martin DL: Two isoforms of glutamate decarboxylase: Why? Trends Pharmacol Sci 1998;19:500–505.
  62. Martin DL, Tobin AJ: Mechanisms controlling GABA synthesis and degradation in the brain; in Martin DL, Olsen RW (eds): GABA in the Nervous System: The View at Fifty Years. Philadelphia, Lippincott, Williams & Wilkins, 2000, pp 25–41.
  63. Sun D, Murali SG: Na+-K+-2Cl cotransporter in immature cortical neurons: A role in intracellular Cl regulation. J Neurophysiol 1999;81:1939–1948.
  64. Delpire E: Cation-chloride cotransporters in neuronal communication. News Physiol Sci 2000;15:309–312.
  65. Sung KW, Kirby M, McDonald MP, Lovinger DM, Delpire E: Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice. J Neurosci 2000;20:7531–7538.
  66. Plotkin MD, Kaplan MR, Peterson LN, Gullans SR, Hebert SC, Delpire E: Expression of the Na+-K+-2Cl cotransporter BSC2 in the nervous system. Am J Physiol 1997;272:C173–183.
  67. Yan Y, Dempsey RJ, Sun D: Expression of Na+-K+-Cl cotransporter in rat brain during development and its localization in mature astrocytes. Brain Res 2001;911:43–55.
  68. Marty S, Wehrle R, Alvarez-Leefmans FJ, Gasnier B, Sotelo C: Postnatal maturation of Na+, K+, 2Cl cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: An immunocytochemical analysis. Eur J Neurosci 2002;15:233–245.
  69. Hara M, Inoue M, Yasukura T, Ohnishi S, Mikami Y, Inagaki C: Uneven distribution of intracellular Cl in rat hippocampal neurons. Neurosci Lett 1992;143:135–138.
  70. Nusser Z, Sieghart W, Benke D, Fritschy JM, Somogyi P: Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells. Proc Natl Acad Sci USA 1996;93:11939–11944.
  71. Wisden W, Laurie DJ, Monyer H, Seeburg PH: The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci 1992;12:1040–1062.
  72. Weiland NG, Orchinik M: Specific subunit mRNAs of the GABAA receptor are regulated by progesterone in subfields of the hippocampus. Mol Brain Res 1995;32:271–278.
  73. McCarthy MM, Kaufman LC, Brooks PJ, Pfaff DW, Schwartz-Giblin S: Estrogen modulation of mRNA levels for the two forms of glutamic acid decarboxylase (GAD) in female rat brain. J Comp Neurol 1995;360:685–697.
  74. Curran-Rauhut MA, Petersen SL: Regulation of glutamic acid decarboxylase 65 and 67 gene expression by ovarian steroids: Identification of two functionally distinct populations of GABA neurones in the preoptic area. J Neuroendocrinol 2002;14:310–317.
  75. Asada H, Kawamura Y, Maruyama K, Kume H, Ding R, Ji FY, Kanbara N, Kuzume H, Sanbo M, Yagi T, Obata K: Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures. Biochem Biophys Res Commun 1996;229:891–895.
  76. Asada H, Kawamura Y, Maruyama K, Kume H, Ding RG, Kanbara N, Kuzume H, Sanbo M, Yagi T, Obata K: Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proc Natl Acad Sci USA 1997;94:6496–6499.
  77. Petersen SL, Carpenter CD: Studies of glutamic acid decarboxylase (GAD) promoter regulation by estradiol (E2) and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD). Abstr Soc Neurosci 2002;863:3.
  78. Russell JM: Sodium-potassium-chloride cotransport. Physiol Rev 2000;80:211–276.
  79. Wang H, Yan Y, Kintner DB, Lytle C, Sun D: GABA-mediated trophic effect on oligodendrocytes requires Na-K-2Cl cotransport activity. J Neurophysiol 2003;90:1257–1265.
  80. Margeta-Mitrovic M, Mitrovic I, Riley RC, Jan LY, Basbaum AI: Immunohistochemical localization of GABAB receptors in the rat central nervous system. J Comp Neurol 1999;405:299–321.
  81. Sloviter RS, Ali-Akbarian L, Elliott RC, Bowery BJ, Bowery NG: Localization of GABAB (R1) receptors in the rat hippocampus by immunocytochemistry and high resolution autoradiography, with specific reference to its localization in identified hippocampal interneuron subpopulations. Neuropharmacology 1999;38:1707–1721.
  82. Velisek L, Veliskova J: Estrogen treatment protects GABAB inhibition in the dentate gyrus of female rats after kainic acid-induced status epilepticus. Epilepsia 2002;43S5:146–151.
  83. Mobbs CV, Gee DM, Finch CE: Reproductive senescence in female C57BL/6J mice: Ovarian impairments and neuroendocrine impairments that are partially reversible and delayable by ovariectomy. Endocrinology 1984;115:1653–1662.
  84. Adams MM, Oung T, Morrison JH, Gore AC: Length of postovariectomy interval and age, but not estrogen replacement, regulate N-methyl-D-aspartate receptor mRNA levels in the hippocampus of female rats. Exp Neurol 2001;170:345–356.
  85. Gee DM, Flurkey K, Mobbs CV, Sinha YN, Finch CE: The regulation of luteinizing hormone and prolactin in C57BL/6J mice: Effects of estradiol implant size, duration of ovariectomy, and aging. Endocrinology 1984;114:685–693.
  86. Yuri K, Kawata M: Estrogen receptor-immunoreactive neurons contain calcitonin gene-related peptide, methionine-enkephalin or tyrosine hydroxylase in the female rat preoptic area. Neurosci Res 1994;21:135–141.
  87. Funabashi T, Kleopoulos SP, Brooks PJ, Kimura F, Pfaff DW, Shinohara K, Mobbs CV: Changes in estrogenic regulation of estrogen receptor alpha mRNA and progesterone receptor mRNA in the female rat hypothalamus during aging: An in situ hybridization study. Neurosci Res 2000;38:85–92.
  88. Caba M, Beyer C, Gonzalez-Mariscal G, Morrell JI: Immunocytochemical detection of estrogen receptor-alpha in the female rabbit forebrain: topography and regulation by estradiol. Neuroendocrinology 2003;77:208–222.
  89. Adams MM, Fink SE, Shah RA, Janssen WG, Hayashi S, Milner TA, McEwen BS, Morrison JH: Estrogen and aging affect the subcellular distribution of estrogen receptor-alpha in the hippocampus of female rats. J Neurosci 2002;22:3608–3614.

Pay-per-View Options
Direct payment This item at the regular price: USD 38.00
Payment from account With a Karger Pay-per-View account (down payment USD 150) you profit from a special rate for this and other single items.
This item at the discounted price: USD 26.50