Neurochemical Research

, Volume 35, Issue 11, pp 1787–1795 | Cite as

Na+, K+ ATPase Activity Is Reduced in Amygdala of Rats with Chronic Stress-Induced Anxiety-Like Behavior

  • Leonardo Crema
  • Michele Schlabitz
  • Bárbara Tagliari
  • Aline Cunha
  • Fabrício Simão
  • Rachel Krolow
  • Letícia Pettenuzzo
  • Christianne Salbego
  • Deusa Vendite
  • Angela T. S. Wyse
  • Carla Dalmaz


In this study, we examined the effects of two chronic stress regimens upon anxiety-like behavior, Na+, K+-ATPase activity and immunocontent, and oxidative stress parameters (antioxidant enzymes and reactive oxygen species production) in the amygdala. Male rats were subjected to chronic unpredictable and to chronic restraint stress for 40 days. Subsequently, anxiety-like behavior was examined. Both stressed groups presented increased anxiety-like behavior. Reduced amygdalal Na+, K+-ATPase activity in the synaptic plasma membranes was also observed, without alterations in the amygdala immunocontent. In addition, when analyzing oxidative stress parameters, only superoxide dismutase activity was decreased in the amygdala of animals subjected to unpredictable stress. We conclude that both models of chronic stress lead to anxiety-like behavior and decreased amygdalal Na+, K+-ATPase activity, which appears not to be related to oxidative imbalance. The relationship between this decreased activity and anxiety-like behavior remains to be studied.


Chronic stress Depression Anxiety Amygdala Na+, K+-ATPase Oxidative stress 



This work was supported by the National Research Council of Brazil (CNPq), and FINEP/Rede IBN 01.06.0842-00. Leonardo M. Crema was the recipient of a CNPq fellowship.


  1. 1.
    Ericinska M, Silver IA (1994) Ions and energy in mammalian brain. Prog Neurobiol 16:37–71CrossRefGoogle Scholar
  2. 2.
    Shull GE, Greeb J, Lingrel JB (1986) Molecular cloning of three distinct forms of the Na+, K+-ATPase alpha-subunit from rat brain. Biochemistry 25:8125–8132CrossRefPubMedGoogle Scholar
  3. 3.
    Jewell EA, Shamraj OI, Lingrel JB (1992) Isoforms of the alpha subunit of Na, K-ATPase and their significance. Acta Physiol Scand Suppl 607:161–169PubMedGoogle Scholar
  4. 4.
    Segall L, Daly SE, Blostein R (2001) Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na, K-ATPase. J Biol Chem 276:31535–31541CrossRefPubMedGoogle Scholar
  5. 5.
    Streck EL, Zugno AI, Tagliari B et al (2001) Inhibition of rat brain Na+, K+-ATPase activity induced by homocysteine is probably mediated by oxidative stress. Neurochem Res 26:1195–1200CrossRefPubMedGoogle Scholar
  6. 6.
    Wilhelm EA, Jesse CR, Bortolatto CF et al (2009) Anticonvulsant and antioxidant effects of 3-alkynyl selenophene in 21-day-old rats on pilocarpine model of seizures. Brain Res Bull 79:281–287CrossRefPubMedGoogle Scholar
  7. 7.
    Morel P, Tallineau C, Pontcharraud R et al (1998) Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ATPase in rat striatal synaptosomes. Neurochem Int 33:531–540CrossRefPubMedGoogle Scholar
  8. 8.
    Petrushanko I, Bogdanov N, Bulygina E et al (2006) Na-K-ATPase in rat cerebellar granule cells is redox sensitive. Am J Physiol Regul Integr Comp Physiol 290:R916–R925PubMedGoogle Scholar
  9. 9.
    Wang P, Zeng T, Zhang CL et al (2009) Lipid peroxidation was involved in the memory impairment of carbon monoxide-induced delayed neuron damage. Neurochem Res 34:1293–1298CrossRefPubMedGoogle Scholar
  10. 10.
    Long J, Liu C, Sun L et al (2009) Neuronal mitochondrial toxicity of malondialdehyde: inhibitory effects on respiratory function and enzyme activities in rat brain mitochondria. Neurochem Res 34:786–794CrossRefPubMedGoogle Scholar
  11. 11.
    Choi IY, Yan H, Park YK et al (2009) Sauchinone reduces oxygen-glucose deprivation-evoked neuronal cell death via suppression of intracellular radical production. Arch Pharm Res 32:1599–1606CrossRefPubMedGoogle Scholar
  12. 12.
    Cochrane CG (1991) Mechanisms of oxidant injury of cells. Mol Aspects Med 12:137–147CrossRefPubMedGoogle Scholar
  13. 13.
    Metodiewa D, Koska C (2000) Reactive oxygen species and reactive nitrogen species: relevance to cyto(neuro)toxic events and neurologic disorders. An overview. Neurotoxicity Res 1:197–233CrossRefGoogle Scholar
  14. 14.
    Olanow CW (1992) An introduction to the free radical hypothesis in Parkinson’s disease. Ann Neurol 32(Suppl):S2–S9CrossRefPubMedGoogle Scholar
  15. 15.
    Halliwell B, Gutteridge JMC (2007) Free radicals in biology and medicine, 4a edn. Oxford University Press, OxfordGoogle Scholar
  16. 16.
    Wyse ATS, Streck EL, Worm P et al (2000) Preconditioning prevents the inhibition of Na+, K+ ATPase activity after brain ischemia. Neurochem Res 25:969–973Google Scholar
  17. 17.
    Grisar T (1984) Glial and neuronal Na+, K+ pump in epilepsy. Ann Neurol 16:S128–S134CrossRefPubMedGoogle Scholar
  18. 18.
    Pisani A, Martella G, Tscherter A et al (2006) Enhanced sensitivity of DJ-1-deficient dopaminergic neurons to energy metabolism impairment: role of Na+/K+ ATPase. Neurobiol Dis 23:54–60CrossRefPubMedGoogle Scholar
  19. 19.
    Hattori N, Kitagawa K, Higashida T et al (1998) CI-ATPase and Na+/K(+)-ATPase activities in Alzheimer’s disease brains. Neurosci Lett 254:141–144CrossRefPubMedGoogle Scholar
  20. 20.
    Yu SP (2003) Na+, K+ ATPase: the new face of an old player in pathogenesis and apoptotic/hybrid cell death. Biochem Pharmacol 66:1601–1609CrossRefPubMedGoogle Scholar
  21. 21.
    Hokin-Neaverson M, Jefferson JW (1989) Erythrocytes sodium pump activity in bipolar affective disorder and other psychiatry disorders. Neuropsychobiology 22:1–7CrossRefPubMedGoogle Scholar
  22. 22.
    Mynett-Johnson L, Murphy V, McCormack J et al (1998) Evidence for an allelic association between bipolar disorder and a Na+, K+ adenosine triphosphatase alpha subunit gene (ATP1A3). Biol Psychiatry 44:47–51CrossRefPubMedGoogle Scholar
  23. 23.
    Rybakowsky J, Potok E, Strzizewski W et al (1984) Erythrocyte cation transport disturbances in patients with endogenous depression. Clinical Experim Pharmacol Phys 11:319–326CrossRefGoogle Scholar
  24. 24.
    Wood AJ, Smith CE, Clarke EE et al (1991) Altered in vitro adaptative responses of lymphocyte Na, K-ATPase in patients with manic depressive psychosis. J Affect Disord 21:199–206CrossRefPubMedGoogle Scholar
  25. 25.
    EI-Mallakh RS, Wyatt RJ (1995) The Na+, K+ ATPase hypothesis for bipolar illness. Biol Psychiatry 37:235–244CrossRefGoogle Scholar
  26. 26.
    Riegel RE, Valvassori SS, Elias G et al (2009) Animal model of mania induced by ouabain: evidence of oxidative stress in submitochondrial particles of the rat brain. Neurochem Int 55:491–495CrossRefPubMedGoogle Scholar
  27. 27.
    Gamaro GD, Streck EL, Matté C et al (2003) Reduction of hippocampal Na+, K+ ATPase activity in rats subjected to an experimental model of depression. Neurochem Res 28:1339–1344CrossRefPubMedGoogle Scholar
  28. 28.
    de Vasconcellos AP, Tabajara AS, Ferrari C et al (2003) Effect of chronic stress on spatial memory in rats is attenuated by lithium treatment. Physiol Behav 79:143–149CrossRefPubMedGoogle Scholar
  29. 29.
    Pucilowski O, Overstreet DH, Rezvani AH et al (1993) Chronic mild stress-induced anhedonia: greater effectin a genetic rat model of depression. Physiol Behav 54:1215–1220CrossRefPubMedGoogle Scholar
  30. 30.
    Willner P (1991) Animal models as simulations of depression. TIPS 12:131–136PubMedGoogle Scholar
  31. 31.
    D’Aquila PS, Brain P, Willner P (1994) Effects of chronic mild stress on performance in behavioural tests relevant to anxiety and depression. Physiol Behav 56:861–867CrossRefPubMedGoogle Scholar
  32. 32.
    Ely DR, Dapper V, Marasca J et al (1997) Effect of restraint stress on feeding behavior of rats. Physiol Behav 61:395–398CrossRefPubMedGoogle Scholar
  33. 33.
    Torres IL, Gamaro GD, Silveira-Cucco SN et al (2001) Effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices. Braz J Med Biol Res 34:111–116PubMedGoogle Scholar
  34. 34.
    Martí O, Armario A (1997) Influence of regularity of exposure to chronic stress on the pattern of habituation of pituitary-adrenal hormones, prolactin and glucose. Stress 1:179–189CrossRefPubMedGoogle Scholar
  35. 35.
    Sibille E, Wang Y, Joeyen-Waldorf J et al (2009) A molecular signature of depression in the amygdala. Am J Psychiatry 166:1011–1024CrossRefPubMedGoogle Scholar
  36. 36.
    Yang TT, Simmons AN, Matthews SC et al (2010) Adolescents with major depression demonstrate increased amygdala activation. J Am Acad Child Adolesc Psychiatry 49:42–51CrossRefPubMedGoogle Scholar
  37. 37.
    LeDoux J (2007) The amygdala. Curr Biol 17:R868–R874CrossRefPubMedGoogle Scholar
  38. 38.
    Kim MJ, Whalen PJ (2009) The structural integrity of an amygdala-prefrontal pathway predicts trait anxiety. J Neurosci 29:11614–11618CrossRefPubMedGoogle Scholar
  39. 39.
    Goldin PR, Manber-Ball T, Werner K et al (2009) Neural mechanisms of cognitive reappraisal of negative self-beliefs in social anxiety disorder. Biol Psychiatry 66:1091–1099CrossRefPubMedGoogle Scholar
  40. 40.
    Wolfensberger SP, Veltman DJ, Hoogendijk WJ et al (2008) Amygdala responses to emotional faces in twins discordant or concordant for the risk for anxiety and depression. Neuroimage 41:544–552CrossRefPubMedGoogle Scholar
  41. 41.
    Ikeda K, Onaka T, Yamakado M et al (2003) Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump alpha2 subunit (Atp1a2)-deficient mice. J Neurosci 23:4667–4676PubMedGoogle Scholar
  42. 42.
    Moseley AE, Williams MT, Schaefer TL et al (2007) Deficiency in Na+, K+ ATPase alpha isoform genes alters spatial learning, motor activity, and anxiety in mice. J Neurosci 27:616–626CrossRefPubMedGoogle Scholar
  43. 43.
    Johnson LR, Farb C, Morrison JH et al (2005) Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala. Neuroscience 136:289–299CrossRefPubMedGoogle Scholar
  44. 44.
    Rossie S, Jayachandran H, Meisel RL (2006) Cellular co-localization of protein phosphatase 5 and glucocorticoid receptors in rat brain. Brain Res 1111:1–11CrossRefPubMedGoogle Scholar
  45. 45.
    Silveira PP, Portella AK, Clemente Z et al (2005) The effect of neonatal handling on adult feeding behavior is not an anxiety-like behavior. Int J Dev Neurosci 23:93–99CrossRefPubMedGoogle Scholar
  46. 46.
    Prut L, Belzung C (2003) The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3–33CrossRefPubMedGoogle Scholar
  47. 47.
    Jones DH, Matus AI (1974) Isolation of plasma synaptic membrane from brain by combination flotation-sedimentation density gradient centrifugation. Biochim Biophys Acta 356:276–287CrossRefPubMedGoogle Scholar
  48. 48.
    Chan KM, Delfer D, Junger KD (1986) A direct colorimetric assay for Ca2+ -stimulated ATPase activity. Anal Biochem 157:375–380CrossRefPubMedGoogle Scholar
  49. 49.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-die-binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  50. 50.
    Delmas-Beauvieux MC, Peuchant E, Dumon MF et al (1995) Relationship between red blood cell antioxidant enzymatic system status and lipoperoxidation during the acute phase of malaria. Clin Biochem 28:163–169CrossRefPubMedGoogle Scholar
  51. 51.
    Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefPubMedGoogle Scholar
  52. 52.
    Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77:325–333CrossRefPubMedGoogle Scholar
  53. 53.
    Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by a dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27:612–616CrossRefPubMedGoogle Scholar
  54. 54.
    Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  55. 55.
    Katz RJ, Roth KA, Carroll BJ (1981) Acute and chronic stress effects on open field activity in the rat: Implications for a model of depression. Neurosci Biobehav Rev 5:247–251CrossRefPubMedGoogle Scholar
  56. 56.
    Gamaro GD, Manoli LP, Torres IL et al (2003) Effects of chronic variate stress on feeding behavior and on monoamine levels in different rat brain structures. Neurochem Int 42:107–114CrossRefPubMedGoogle Scholar
  57. 57.
    Silveira PP, Xavier MH, Souza FH et al (2000) Interaction between repeated restraint stress and concomitant midazolam administration on sweet food ingestion in rats. Braz J Med Biol Res 33:1343–1350CrossRefPubMedGoogle Scholar
  58. 58.
    Alves R, Barbosa de Carvalho JG, Benedito MA (2005) High and low rearing subgroups of rats selected in the open field differ in the activity of K+ -stimulated p-nitrophenylphosphatase in the hippocampus. Brain Res 1058:178–182CrossRefPubMedGoogle Scholar
  59. 59.
    Mineur YS, Belzung C, Crusio WE (2006) Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behav Brain Res 175:43–50CrossRefPubMedGoogle Scholar
  60. 60.
    Joo Y, Choi KM, Lee YH et al (2009) Chronic immobilization stress induces anxiety- and depression-like behaviors and decreases transthyretin in the mouse cortex. Neurosci Lett 461:121–125CrossRefPubMedGoogle Scholar
  61. 61.
    Noschang CG, Pettenuzzo LF, Toigo EV et al (2009) Sex-specific differences on caffeine consumption and chronic stress-induced anxiety-like behavior and DNA breaks in the hippocampus. Pharmacol Biochem Behav 94:63–69CrossRefPubMedGoogle Scholar
  62. 62.
    Rodrigues SM, LeDoux JE, Sapolsky RM (2009) The influence of stress hormones on fear circuitry. Annu Rev Neurosci 32:289–313CrossRefPubMedGoogle Scholar
  63. 63.
    Vyas A, Mitra R, Shankaranarayana Rao BS et al (2002) Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. J Neurosci 22:6810–6818PubMedGoogle Scholar
  64. 64.
    Vyas A, Jadhav S, Chattarji S (2006) Prolonged behavioral stress enhances synaptic connectivity in the basolateral amydala. Neuroscience 143:387–393CrossRefPubMedGoogle Scholar
  65. 65.
    de Vasconcellos AP, Zugno AI, Dos Santos AH et al (2005) Na+, K(+)-ATPase activity is reduced in hippocampus of rats submitted to an experimental model of depression: effect of chronic lithium treatment and possible involvement in learning deficits. Neurobiol Learn Mem 84:102–110CrossRefPubMedGoogle Scholar
  66. 66.
    Ulrich-Lai YM, Herman JP (2009) Neural regulation of endocrine and autonomic stress responses. Nat Rev Neurosci 10:397–409CrossRefPubMedGoogle Scholar
  67. 67.
    Westenberg HG (2009) Recent advances in understanding and treating social anxiety disorder. CNS Spectr 14(2 Suppl 3):24–33PubMedGoogle Scholar
  68. 68.
    Papaleo F, Crawley JN, Song J et al (2008) Genetic dissection of the role of catechol-O-methyltransferase in cognition and stress reactivity in mice. J Neurosci 28:8709–8723CrossRefPubMedGoogle Scholar
  69. 69.
    Paul C, Schöberl F, Weinmeister P et al (2008) Signaling through cGMP-dependent protein kinase I in the amygdala is critical for auditory-cued fear memory and long-term potentiation. J Neurosci 28:14202–14212CrossRefPubMedGoogle Scholar
  70. 70.
    Farman N, Bonvalet JP, Seckl JR (1994) Aldosterone selectively increases Na+, K+ ATPase alpha 3-subunit mRNA expression in rat hippocampus. Am J Physiol 266(2 Pt 1):C423–C428PubMedGoogle Scholar
  71. 71.
    Awaiss D, Shao Y, Isamil-Beigi F (2000) Thyroid hormone regulation of myocardial Na+, K+ ATPase gene expression. J Mol Cell Cardiol 32:1969–1980CrossRefGoogle Scholar
  72. 72.
    Hernandez RJ (1992) Na+, K+ ATPase regulation by neurotransmitters. Neurochem Int 20:1–10CrossRefGoogle Scholar
  73. 73.
    Swann AC (1983) Stimulation of brain Na+, K+ ATPase by norepinephrine in vivo: prevention by receptor antagonists and enhancement by repeated stimulation. Brain Res 260:338–341CrossRefPubMedGoogle Scholar
  74. 74.
    Peña-Rangel MT, Rosalio MC, Hernandez-Rodriguez J (1999) Regulation of glial Na+, K+ ATPase by serotonin: identification of participating receptors. Neurochem Res 24:643–649CrossRefPubMedGoogle Scholar
  75. 75.
    Rose EM, Koo JC, Antflick JE et al (2009) Glutamate transporter coupling to Na, K-ATPase. J Neurosci 29:8143–8155CrossRefPubMedGoogle Scholar
  76. 76.
    Dobretsov M, Stimers JR (2005) Neuronal function and alpha3 isoform of the Na/K-ATPase. Front Biosci 10:2373–2396CrossRefPubMedGoogle Scholar
  77. 77.
    Taguchi K, Kumanogoh H, Nakamura S et al (2007) Ouabain-induced isoform-specific localization change of the Na+, K+ -ATPase alpha subunit in the synaptic plasma membrane of rat brain. Neurosci Lett 413:42–45CrossRefPubMedGoogle Scholar
  78. 78.
    Grillo C, Piroli G, González SL et al (1994) Glucocorticoid regulation of mRNA encoding (Na + K) ATPase alpha 3 and beta 1 subunits in rat brain measured by in situ hybridization. Brain Res 657:83–91CrossRefPubMedGoogle Scholar
  79. 79.
    Hamid H, Gao Y, Lei Z et al (2009) Effect of ouabain on sodium pump alpha-isoform expression in an animal model of mania. Prog Neuropsychopharmacol Biol Psychiatry 33:1103–1106CrossRefPubMedGoogle Scholar
  80. 80.
    de Carvalho Aguiar P, Sweadner KJ, Penniston JT et al (2004) Mutations in the Na+/K+ -ATPase alpha3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism. Neuron 43:169–175CrossRefPubMedGoogle Scholar
  81. 81.
    Moseley AE, Lieske SP, Wetzel RK et al (2003) The Na, K-ATPase alpha 2 isoform is expressed in neurons, and its absence disrupts neuronal activity in newborn mice. J Biol Chem 278:5317–5324CrossRefPubMedGoogle Scholar
  82. 82.
    Martín-Vasallo P, Wetzel RK, García-Segura LM et al (2000) Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na, K-ATPase. Glia 31:206–218CrossRefPubMedGoogle Scholar
  83. 83.
    Sweadner KJ (1992) Overlapping and diverse distribution of Na-K ATPase isozymes in neurons and glia. Can J Physiol Pharmacol 70(Suppl):S255–S259PubMedGoogle Scholar
  84. 84.
    Fontella FU, Siqueira IR, Vasconcellos AP et al (2005) Repeated restraint stress induces oxidative damage in rat hippocampus. Neurochem Res 30:105–111CrossRefPubMedGoogle Scholar
  85. 85.
    Liu J, Wang X, Shigenaga MK et al (1996) Immobilization stress causes oxidative damage to lipid, protein, and DNA in the brain of rats. FASEB J 10:1532–1538PubMedGoogle Scholar
  86. 86.
    Namba C, Adachi N, Liu K et al (2002) Suppression of sodium pump activity and an increase in the intracellular Ca2+ concentration by dexamethasone in acidotic mouse brain. Brain Res 957:271–277CrossRefPubMedGoogle Scholar
  87. 87.
    Schoner W (2000) Ouabain, a new steroid hormone of adrenal gland and hypothalamus. Exp Clin Endocrinol Diabetes 108:449–454CrossRefPubMedGoogle Scholar
  88. 88.
    Bauer N, Muller-Ehmsen J, Kramer U et al (2005) Ouabain-like compound changes rapidly on physical exercise in humans and dogs-effects of β-blockade and angiotensin-converting enzyme inhibition. Hypertension 45:1024–1028CrossRefPubMedGoogle Scholar
  89. 89.
    Kölbel F, Schreiber V (1996) The endogenous digitalis-like factor. Mol Cell Biochem 160–161:111–115CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Leonardo Crema
    • 1
    • 2
  • Michele Schlabitz
    • 1
  • Bárbara Tagliari
    • 1
    • 3
  • Aline Cunha
    • 1
  • Fabrício Simão
    • 1
    • 3
  • Rachel Krolow
    • 1
    • 3
  • Letícia Pettenuzzo
    • 1
    • 3
  • Christianne Salbego
    • 1
    • 3
  • Deusa Vendite
    • 1
    • 2
  • Angela T. S. Wyse
    • 1
    • 3
  • Carla Dalmaz
    • 1
    • 2
    • 3
  1. 1.Departamento de Bioquímica, ICBSUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  2. 2.Programa de Pós-Graduação em Neurociências, ICBSUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  3. 3.Programa de Pós-Graduação em Bioquímica, ICBSUniversidade Federal do Rio Grande do SulPorto AlegreBrazil

Personalised recommendations