Abstract
Mild hyperhomocysteinemia is a risk factor for psychiatric and neurodegenerative diseases, whose mechanisms between them are not well-known. In the present study, we evaluated the emotional behavior and neurochemical pathways (ATPases, glutamate homeostasis, and cell viability) in amygdala and prefrontal cortex rats subjected to mild hyperhomocysteinemia (in vivo studies). The ex vivo effect of homocysteine on ATPases and redox status, as well as on NMDAR antagonism by MK-801 in same structures slices were also performed. Wistar male rats received a subcutaneous injection of 0.03 µmol Homocysteine/g of body weight or saline, twice a day from 30 to 60th–67th days of life. Hyperhomocysteinemia increased anxiety-like behavior and tended to alter locomotion/exploration of rats, whereas sucrose preference and forced swimming tests were not altered. Glutamate uptake was not changed, but the activities of glutamine synthetase and ATPases were increased. Cell viability was not altered. Ex vivo studies (slices) showed that homocysteine altered ATPases and redox status and that MK801, an NMDAR antagonist, protected amygdala (partially) and prefrontal cortex (totally) effects. Taken together, data showed that mild hyperhomocysteinemia impairs the emotional behavior, which may be associated with changes in ATPase and glutamate homeostasis, including glutamine synthetase and NMDAR overstimulation that could lead to excitotoxicity. These findings may be associated with the homocysteine risk factor on psychiatric disorders development and neurodegeneration.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- ATP:
-
Adenine triphosphate
- ATPase:
-
Adenosine triphosphatase enzyme
- DNA:
-
Deoxyribonucleic acid
- GLAST:
-
Glutamate aspartate transporter
- GLT-1:
-
Glutamate transporter 1
- HCY:
-
Homocysteine
- HHCY:
-
Hyperhomocysteinemia
References
Arteni NS, Pereira LO, Rodrigues AL et al (2010) Lateralized and sex-dependent behavioral and morphological effects of unilateral neonatal cerebral hypoxia-ischemia in the rat. Behav Brain Res 210:92–98. https://doi.org/10.1016/j.bbr.2010.02.015
Bailey KR, Crawley JN (2009) Anxiety-Related Behaviors in Mice. In: Buccafusco JJ (ed) Methods of Behavior Analysis in Neuroscience., 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis, Boca Raton (FL)
Baird L, Dinkova-Kostova AT (2011) The cytoprotective role of the Keap1-Nrf2 pathway. Arch Toxicol 85:241–272. https://doi.org/10.1007/s00204-011-0674-5
Bandelow B, Michaelis S (2015) Epidemiology of anxiety disorders in the 21st century. Dialogues Clin Neurosci 17:327–335
Belleau EL, Pedersen WS, Miskovich TA et al (2018) Cortico-limbic connectivity changes following fear extinction and relationships with trait anxiety. Soc Cogn Affect Neurosci 13:1037–1046. https://doi.org/10.1093/scan/nsy073
Bhatia P, Singh N (2015) Homocysteine excess: Delineating the possible mechanism of neurotoxicity and depression. Fundam Clin Pharmacol 29:522–528. https://doi.org/10.1111/fcp.12145
Blanke ML, Vandongen AMJ (2009) Activation Mecanisms of the NMDA Receptor. In: Van Dongen A (ed) Biology of the NMDA Receptor. CRC Press/Taylor & Francis, Boca Raton (FL), pp 1–24
Boldyrev AA (2005) Homocysteinic acid causes oxidative stress in lymphocytes by potentiating toxic effect of NMDA. Bull Exp Biol Med 140:33–37. https://doi.org/10.1007/s10517-005-0404-1
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Browne RW, Armstrong D (1998) Reduced glutathione and glutathione disulfide. Methods Mol Biol 108:347–352
Carageorgiou H, Sideris AC, Messari I et al (2008) The effects of rivastigmine plus selegiline on brain acetylcholinesterase, (Na+, K+)-, Mg2+- ATPase activities, antioxidant status, and learning performance of aged rats. Neuropsychiatr Dis Treat 4:687–699
Chan K, Delfert D, Junger K (1986) A direct colorimetric assay for Ca2+-stimulated ATPase activity. Anal Biochem 157:375–380
Choudhury S, Borah A (2015) Activation of NMDA receptor by elevated homocysteine in chronic liver disease contributes to encephalopathy. Med Hypotheses 85:64–67. https://doi.org/10.1016/j.mehy.2015.03.027
Chung K, Chiou H, Chen Y (2017) Associations between serum homocysteine levels and anxiety and depression among children and adolescents in Taiwan. Sci Rep. https://doi.org/10.1038/s41598-017-08568-9
Clarke R (2011) Homocysteine, B vitamins, and the risk of cardiovascular disease. Clin Chem 57:1201–1202. https://doi.org/10.1373/clinchem.2011.164855
Crema L, Schlabitz M, Tagliari B et al (2010) Na+, K+-ATPase activity is reduced in Amygdala of rats with chronic stress-induced anxiety-like behavior. Neurochem Res 35:1787–1795. https://doi.org/10.1007/s11064-010-0245-9
da Silva FF, de Ferreira AP, O, Ribeiro LR, et al (2016) The impact of previous physical training on redox signaling after traumatic brain injury in rats: A behavioral and neurochemical approach. J Neurotrauma 33:1317–1330. https://doi.org/10.1089/neu.2015.4068
de Arnaiz GR, L, Ordieres MGL, (2014) Brain Na+, K+-ATPase activity in aging and disease. Int J Biomed Sci 10:85–102
de Wyse AT, S, Streck EL, Worm P, et al (2000) Preconditioning prevents the inhibition of NA+, K+-ATPase activity after brain ischemia. Neurochem Res 25:971–975. https://doi.org/10.1023/A:1007504525301
Diehl LA, Pereira NDSC, Laureano DP et al (2014) Contextual fear conditioning in maternal separated rats: The amygdala as a site for alterations. Neurochem Res 39:384–393. https://doi.org/10.1007/s11064-013-1230-x
dos Santos AQ, Nardin P, Funchal C et al (2006) Resveratrol increases glutamate uptake and glutamine synthetase activity in C6 glioma cells. Arch Biochem Biophys 453:161–167. https://doi.org/10.1016/j.abb.2006.06.025
dos Santos TM, Siebert C, Federizzi M et al (2019) Chronic mild Hyperhomocysteinemia impairs energy metabolism, promotes DNA damage and induces a Nrf2 response to oxidative stress in rats brain. Cell Mol Neurobiol. https://doi.org/10.1007/s10571-019-00674-8
dos Santos TM, Kolling J, Siebert C et al (2017) Effects of previous physical exercise to chronic stress on long-term aversive memory and oxidative stress in amygdala and hippocampus of rats. Int J Dev Neurosci 56:58–67. https://doi.org/10.1016/j.ijdevneu.2016.12.003
Esnafoglu E, Yaman E (2017) Vitamin B12, folic acid, homocysteine and vitamin D levels in children and adolescents with obsessive compulsive disorder. Psychiatry Res 254:232–237. https://doi.org/10.1016/j.psychres.2017.04.032
Finkelstein JD (2007) Metabolic regulatory properties of S-adenosylmethionine and S-adenosylhomocysteine. Clin Chem Lab Med 45:1694–1699. https://doi.org/10.1515/CCLM.2007.341
Foo K, Blumenthal L, Man HY (2012) Regulation of neuronal bioenergy homeostasis by glutamate. Neurochem Int 61:389–396. https://doi.org/10.1016/j.neuint.2012.06.003
Ganapathy PS, White RE, Ha Y et al (2011) The role of N-methyl-D-aspartate receptor activation in homocysteine-induced death of retinal ganglion cells. Investig Ophthalmol vis Sci 52:5515–5524. https://doi.org/10.1167/iovs.10-6870
Green LC, Wagner DA, Glogowski J et al (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126:131–138. https://doi.org/10.1016/0003-2697(82)90118-X
Grigor’yan GA, Gulyaeva N V, (2017) Modeling depression in animals : Behavior as the basis for the methodology, assessment criteria, and classification. Neurosci Behav Physiol 47:204–216. https://doi.org/10.1007/s11055-016-0386-7
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 23:4667–4676
Izquierdo I, Furini CRG, Myskiw JC (2016) Fear memory. Physiol Rev 96:695–750. https://doi.org/10.1152/physrev.00018.2015
Jendricko T, Andelko V, Grubisic-llic M et al (2009) Progress in Neuro-Psychopharmacology & Biological Psychiatry Homocysteine and serum lipids concentration in male war veterans with posttraumatic stress disorder. Prog Neuro-Psychopharmacology Biol Psychiatry 33(33):134–140. https://doi.org/10.1016/j.pnpbp.2008.11.002
John CS, Sypek EI, Carlezon WA et al (2015) Blockade of the GLT-1 transporter in the central nucleus of the amygdala induces both anxiety and depressive-like symptoms. Neuropsychopharmacol 40:1700–1708. https://doi.org/10.1038/npp.2015.16
Kim HK, Nunes PV, Oliveira KC et al (2016) Neuropathological relationship between major depression and dementia: A hypothetical model and review. Prog Neuro-Psychopharmacology Biol Psychiatry 67:51–57. https://doi.org/10.1016/j.pnpbp.2016.01.008
Kim MJ, Loucks R, a, Palmer AL, et al (2011) The structural and functional connectivity of the amygdala: From normal emotion to pathological anxiety. Behav Brain Res 223:403–410. https://doi.org/10.1016/j.bbr.2011.04.025
Kinoshita PF, Leite JA, Orellana AMM et al (2016) The influence of Na+, K+-ATPase on glutamate signaling in neurodegenerative diseases and senescence. Front Physiol 7:1–19. https://doi.org/10.3389/fphys.2016.00195
Kumagai A, Sasaki T, Matsuoka K et al (2019) Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption. Synapse 73:e22067. https://doi.org/10.1002/syn.22067
Kumar A, Palfrey HA, Pathak R et al (2017) The metabolism and significance of homocysteine in nutrition and health. Nutr Metab 14:1–12. https://doi.org/10.1186/s12986-017-0233-z
Lavinsky D, Arteni NS, Netto CA (2003) Agmatine induces anxiolysis in the elevated plus maze task in adult rats. Behav Brain Res 141:19–24. https://doi.org/10.1016/s0166-4328(02)00326-1
Lebel CP, Ischiropoulos H (1992) Bondys SC (1992) Evaluation of the probe 2‘,7‘-dichiorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5:227–231
Lewerenz J, Maher P, Maher P (2015) Chronic glutamate toxicity in neurodegenerative diseases — what is the evidence? Front Neurosci 9:1–20. https://doi.org/10.3389/fnins.2015.00469
Li PA, Hou X, Hao S (2017) Mitochondrial biogenesis in neurodegeneration. J Neurosci Res 95:2025–2029. https://doi.org/10.1002/jnr.24042
Liu YY, Zhou XY, Yang LN et al (2017) Social defeat stress causes depression-like behavior with metabolite changes in the prefrontal cortex of rats. PLoS ONE 12:1–16. https://doi.org/10.1371/journal.pone.0176725
Longoni A, Bellaver B, Bobermin LD et al (2017) Homocysteine induces glial reactivity in adult rat astrocyte cultures. Mol Neurobiol 55:1966–1976. https://doi.org/10.1007/s12035-017-0463-0
Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. Readings 193:265–275. https://doi.org/10.1016/0304-3894(92)87011-4
Maguire ME (1988) Magnesium and Cell Proliferation. Ann New York Acad Sci 551:201–217
Mandaviya PR, Stolk L, Heil SG (2014) Homocysteine and DNA methylation : A review of animal and human literature. Mol Genet Metab 113:243–252. https://doi.org/10.1016/j.ymgme.2014.10.006
Marek R, Strobel C, Bredy TW, Sah P (2013) The amygdala and medial prefrontal cortex: Partners in the fear circuit. J Physiol 591:2381–2391. https://doi.org/10.1113/jphysiol.2012.248575
Marklund S (1985) Pyrogallol autoxidation. In: Greenwald RA (ed) Handbook of methods for oxygen radical research, 4th edn. CRC Press, Boca Raton
Marsden WN (2011) Stressor-induced NMDAR dysfunction as a unifying hypothesis for the aetiology, pathogenesis and comorbidity of clinical depression. Med Hypotheses 77:508–528. https://doi.org/10.1016/j.mehy.2011.06.021
Matté C, Mussulini BHMBHM, dos Santos TMTM et al (2010) Hyperhomocysteinemia reduces glutamate uptake in parietal cortex of rats. Int J Dev Neurosci 28:183–187. https://doi.org/10.1016/j.ijdevneu.2009.11.004
McCully KS (2015) Homocysteine metabolism, atherosclerosis, and diseases of aging. Compr Physiol 6:471–505. https://doi.org/10.1002/cphy.c150021
McEwen BS, Eiland L, Hunter RG, Miller MM (2012) Stress and anxiety: Structural plasticity and epigenetic regulation as a consequence of stress. Neuropharmacology 62:3–12. https://doi.org/10.1016/j.neuropharm.2011.07.014
Minagawa H, Watanabe A, Akatsu H et al (2010) Homocysteine, another risk factor for alzheimer disease, impairs apolipoprotein E3 function. J Biol Chem 285:38382–38388. https://doi.org/10.1074/jbc.M110.146258
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 27:616–626. https://doi.org/10.1523/JNEUROSCI.4464-06.2007
Mosmann T (1983) Rapid Colorimetric Assay for Cellular Growth and Survival : Application to Proliferation and Cytotoxicity Assays 65:55–63
Moustafa A, Hewedi D, Eissa A et al (2015) Homocysteine levels in neurologicaL disorders. In: Farooqui T, Farooqui AA (eds) Diet and exercise in cognitive function and neurological diseases. John Wiley & Sons Inc, First Edit, pp 73–81
Moustafa AA, Hewedi DH, Eissa AM et al (2014) Homocysteine levels in schizophrenia and affective disorders — focus on cognition. Front Behav Neurosci 8:1–10. https://doi.org/10.3389/fnbeh.2014.00343
Nielsen CK, Arnt J, Sa C (2000) Intracranial Self-Stimulation and Sucrose Intake Differ as Hedonic Measures following Chronic Mild Stress : Interstrain and Interindividual Differences 107:21–33
Park K, Oh JH, Yoo H et al (2010) Neuroscience Letters ( − ) -Epigallocatechin-3- O -gallate ( EGCG ) reverses caffeine-induced anxiogenic-like effects. Neurosci Lett 481:131–134. https://doi.org/10.1016/j.neulet.2010.06.072
Patki G, Solanki N, Atrooz F et al (2014) Novel mechanistic insights into treadmill exercise based rescue of social defeat-induced anxiety-like behavior and memory impairment in rats. Physiol Behav 130:135–144. https://doi.org/10.1016/j.physbeh.2014.04.011
Paxinos G, Watson C (2007) The Rat Brain in Stereotaxic Coordinates, 6th Editio. Academic Press, San Diego, CA
Pellow S, Chopin P, File SE, Briley M (1985) Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 14:149–167. https://doi.org/10.1016/0165-0270(85)90031-7
Petrea RE, Seshadri S (2008) Homocysteine and neurological disorders. Glutathione Sulfur Amin Acids Hum Heal Dis. https://doi.org/10.1002/9780470475973.ch18
Porsolt RD, Le PM, Jalfre M (1977) Depression : a new animal model sensitive to antidepressant treatments. Nature 266:730–732
Roigé-Castellví J, Murphy M, Fernández-Ballart J, Canals J (2019) Moderately elevated preconception fasting plasma total homocysteine is a risk factor for psychological problems in childhood. Public Health Nutr 22:1615–1623. https://doi.org/10.1017/S1368980018003610
Rubin H (2005) Central Roles of Mg2+ and MgATP2- in the Regulation of Protein Synthesis and Cell Proliferation : Significance for Neoplastic Transformation. Adv Cancer Res 1–58. Doi: https://doi.org/10.1016/S0065-230X(05)93001-7
Sanui H, Rubin H (1982) Changes of intracellular and externally bound cations accompanying serum stimulation of mouse BALB/c 3T3 cells. Exp Cell Res 139:15–25
Scherer EBS, da Cunha AA, Kolling J et al (2011) Development of an animal model for chronic mild hyperhomocysteinemia and its response to oxidative damage. Int J Dev Neurosci 29:693–699. https://doi.org/10.1016/j.ijdevneu.2011.06.004
Scherer EBS, Loureiro SO, Vuaden FC et al (2013) Mild hyperhomocysteinemia reduces the activity and immunocontent, but does not alter the gene expression, of catalytic α subunits of cerebral Na +, K+-ATPase. Mol Cell Biochem 378:91–97. https://doi.org/10.1007/s11010-013-1598-6
Schulkin J (2006) Angst and the amygdala. Dialogues Clin Neurosci 8:407–416. https://doi.org/10.1038/145370a0
Sharma M, Tiwari M, Tiwari RK (2015) Hyperhomocysteinemia: Impact on neurodegenerative diseases. Basic Clin Pharmacol Toxicol 117:287–296. https://doi.org/10.1111/bcpt.12424
Skovierová H, Vidomanová E, Mahmood S et al (2016) The molecular and cellular effect of homocysteine metabolism imbalance on human health. Int J Mol Sci 17:1–18. https://doi.org/10.3390/ijms17101733
Smith K (2014) Mental health: a world of depression. Nature 15:180–181
Srejovic I, Jakovljevic V, Zivkovic V et al (2014) The effects of the modulation of NMDA receptors by homocysteine thiolactone and dizocilpine on cardiodynamics and oxidative stress in isolated rat heart. Mol Cell Biochem 401:97–105. https://doi.org/10.1007/s11010-014-2296-8
Stanger O, Fowler B, Pietrzik K et al (2009) Homocysteine, folate and vitamin B12 in neuropsychiatric diseases: Review and treatment recommendations. Expert Rev Neurother 9:1393–1412. https://doi.org/10.1586/ern.09.75
Stefanello N, Schmatz R, Belmonte L et al (2014) Effects of chlorogenic acid, caffeine, and coffee on behavioral and biochemical parameters of diabetic rats. Mol Cell Biochem 388:277–286. https://doi.org/10.1007/s11010-013-1919-9
Steimer T (2002) The biology of fear- and anxiety-related behaviors. Dialogues Clin Neurosci 4:231–249. https://doi.org/10.1097/ALN.0b013e318212ba87
Suhail M (2010) Na, K-ATPase: Ubiquitous multifunctional transmembrane protein and its relevance to various pathophysiological conditions. J Clin Med Res 2:1–17. https://doi.org/10.4021/jocmr2010.02.263w
Tagliari B, Dos Santos TM, Cunha AA et al (2010) Chronic variable stress induces oxidative stress and decreases butyrylcholinesterase activity in blood of rats. J Neural Transm. https://doi.org/10.1007/s00702-010-0445-0
Tagliari B, Tagliari AP, Schmitz F et al (2011) Chronic variable stress alters inflammatory and cholinergic parameters in hippocampus of rats. Neurochem Res a 36:487–493. https://doi.org/10.1007/s11064-010-0367-0
Walf AA, Frye CA (2007). The Use of the Elevated plus Maze as an Assay of Anxiety-Related Behavior in Rodents. https://doi.org/10.1038/nprot.2007.44
Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77:325–333
Williams KT, Schalinske KL (2010) Homocysteine metabolism and its relation to health and disease. BioFactors 36:19–24. https://doi.org/10.1002/biof.71
Wyse ATS, Bavaresco CS, Reis EA et al (2004) Training in inhibitory avoidance causes a reduction of Na+, K+-ATPase activity in rat hippocampus. Physiol Behav 80:475–479. https://doi.org/10.1016/j.physbeh.2003.10.002
Wyse ATS, Sanches EF, Dos Santos TM et al (2020) Chronic mild hyperhomocysteinemia induces anxiety-like symptoms, aversive memory deficits and hippocampus atrophy in adult rats: New insights into physiopathological mechanisms. Brain Res 1728:146592. https://doi.org/10.1016/J.BRAINRES.2019.146592
Zhou Y, Danbolt NC (2014) Glutamate as a neurotransmitter in the healthy brain. J Neural Transm 121:799–817. https://doi.org/10.1007/s00702-014-1180-8
Acknowledgements
The work has not been published previously and it is not under consideration for publication elsewhere and, if accepted, it will not be published elsewhere in the same form, in English or any other language, including electronically without the written consent of the copyright holder.
Funding
This study was supported by Edital Universal (Proc. 401507/2016)/Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), INCT (EN 465671/2014–4)/Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) – Brazil, and PRONEX (16/2551–0000465-0)/Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS) – Brazil.
Author information
Authors and Affiliations
Contributions
Authors TMS, CS, and Full Professor ATSW (head of the laboratory) developed the experimental model, behavior tests, most biochemical analyzes, statistical analyzes, and manuscript writing. Collaborators LDB and AQS developed assays of glutamate uptake and glutamine synthetase. TMS—Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing—Original Draft, Writing—Review & Editing, Visualization. CS—Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing—Original Draft. LDB—Formal analysis, Investigation, Data Curation. AQ—Formal analysis, Resource, Funding acquisition. ATSW—Conceptualization, Methodology, Validation, Formal analysis, Resources, Writing—Original Draft, Writing—Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest. All authors have approved the final article.
Ethical Approval
Wistar male rats (thirty days old, 70–80 g) were obtained from the Central Animal House of Biochemistry Department, Institute of Basic Health Sciences at the Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil. Animals were maintained with lights on 7:00 a.m. at 7:00 p.m. (12:12 h light/dark cycle) in a colony room constant temperature (22 ± 1 °C), with ad libitum 20% protein commercial chow (w/w) and water (3–4 animals per home cage). The rats were randomized into two groups: control and HCY. Every effort was made to minimize the number of animals and the distress caused throughout the experiment. Official government guidelines for animal care by the Brazilian Federal Law No. 11,794 of October 08, 2008, the NIH Guide to the Care and Use of Laboratory Animals (NIH Publication No. 80–23, 1996), and the ARRIVE guidelines were followed to work development. The experimental protocol was approved by UFRGS Ethics Committee for Animals Use (#33301/2017). The reagents and part of the inputs used in the assays were purchased from Sigma-Aldrich (San Luis, Missouri, EUA) or Merck Millipore (Burlington, Massachusetts, EUA).
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
dos Santos, T.M., Siebert, C., Bobermin, L.D. et al. Mild Hyperhomocysteinemia Causes Anxiety-like Behavior and Brain Hyperactivity in Rodents: Are ATPase and Excitotoxicity by NMDA Receptor Overstimulation Involved in this Effect?. Cell Mol Neurobiol 42, 2697–2714 (2022). https://doi.org/10.1007/s10571-021-01132-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-021-01132-0