Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors


It is known that continuous abuse of amphetamine (AMPH) results in alterations in neuronal structure and cognitive behaviors related to the reward system. However, the impact of AMPH abuse on the hippocampus remains unknown. The aim of this study was to determine the damage caused by AMPH in the hippocampus in an addiction model. We reproduced the AMPH sensitization model proposed by Robinson et al. in 1997 and performed the novel object recognition test (NORt) to evaluate learning and memory behaviors. After the NORt, we performed Golgi–Cox staining, a stereological cell count, immunohistochemistry to determine the presence of GFAP, CASP3, and MT-III, and evaluated oxidative stress in the hippocampus. We found that AMPH treatment generates impairment in short- and long-term memories and a decrease in neuronal density in the CA1 region of the hippocampus. The morphological test showed an increase in the total dendritic length, but a decrease in the number of mature spines in the CA1 region. GFAP labeling increased in the CA1 region and MT-III increased in the CA1 and CA3 regions. Finally, we found a decrease in Zn concentration in the hippocampus after AMPH treatment. An increase in the dopaminergic tone caused by AMPH sensitization generates oxidative stress, neuronal death, and morphological changes in the hippocampus that affect cognitive behaviors like short- and long-term memories.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Experimental design and NORt.
Fig. 2: Neuronal density after 35 days of chronic amphetamine (AMPH) administration.
Fig. 3: Amphetamine (AMPH) sensitization reorganizes the dendritic arbor as well as the dendritic spine dynamics in the pyramidal neurons of the hippocampus.
Fig. 4: Oxidative response in the hippocampus because of the amphetamine (AMPH) sensitization.
Fig. 5: Inflammatory response after amphetamine (AMPH) sensitization.


  1. 1.

    Cao DN, Shi JJ, Hao W, Wu N, Li J. Advances and challenges in pharmacotherapeutics for amphetamine-type stimulants addiction. Eur J Pharm. 2016;780:129–35.

    CAS  Article  Google Scholar 

  2. 2.

    Cadet JL, Bisagno V, Milroy CM. Neuropathology of substance use disorders. Acta Neuropathol. 2014;127:91–107.

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Sulzer D, Sonders MS, Poulsen NW, Galli A. Mechanisms of neurotransmitter release by amphetamines: a review. Prog Neurobiol. 2005;75:406–33.

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    United Nations. World Drug Report 2016. New York: United Nations press; 2016.

  5. 5.

    Robinson TE, Kolb B. Persistent structural modifications in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine. J Neurosci. 1997;17:8491–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Robinson TE, Berridge KC. The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev. 1993;18:247–91.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Vanderschuren LJ, Pierce RC. Sensitization processes in drug addiction. Curr Top Behav Neurosci. 2010;3:179–95.

    PubMed  Article  Google Scholar 

  8. 8.

    Robinson TE, Berridge KC. Review. The incentive sensitization theory of addiction: some current issues. Philos Trans R Soc Lond B Biol Sci. 2008;363:3137–46.

    PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Robinson TE, Berridge KC. Addiction. Annu Rev Psychol. 2003;54:25–53.

    PubMed  Article  Google Scholar 

  10. 10.

    Hedou G, Homberg J, Feldon J, Heidbreder CA. Expression of sensitization to amphetamine and dynamics of dopamine neurotransmission in different laminae of the rat medial prefrontal cortex. Neuropharmacology. 2001;40:366–82.

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Andersen P, Morris R, Amaral DG, Bliss T, O´Keefe J. The hippocampus book. New York: Oxford University Press; 2007.

  12. 12.

    Volkow ND, Wang GJ, Fowler JS, Tomasi D. Addiction circuitry in the human brain. Annu Rev Pharm Toxicol. 2012;52:321–36.

    CAS  Article  Google Scholar 

  13. 13.

    Broadbent NJ, Gaskin S, Squire LR, Clark RE. Object recognition memory and the rodent hippocampus. Learn Mem. 2010;17:5–11.

    PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Rosen ZB, Cheung S, Siegelbaum SA. Midbrain dopamine neurons bidirectionally regulate CA3-CA1 synaptic drive. Nat Neurosci. 2015;18:1763–71.

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    McNamara CG, Dupret D. Two sources of dopamine for the hippocampus. Trends Neurosc. 2017;40:383–4.

    CAS  Article  Google Scholar 

  16. 16.

    Scofield MD, Heinsbroek JA, Gipson CD, Kupchik YM, Spencer S, Smith ACW, et al. The nucleus accumbens: mechanisms of addiction across drug classes reflect the importance of glutamate homeostasis. Pharm Rev. 2016;68:816–71.

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Arroyo-García LE, Vázquez-Roque RA, Díaz A, Treviño S, De La Cruz F, Flores G, et al. The effects of non-selective dopamine receptor activation by apomorphine in the mouse hippocampus. Mol Neurobiol. 2018;55:8625–36.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Arroyo-Garcia LE, Rodríguez-Moreno A, Flores G. Apomorphine effects on the hippocampus. Neural Regeneration Res. 2018;13:2064–6.

    Article  Google Scholar 

  19. 19.

    Cadet JL, Krasnova IN, Jayanthi S, Lyles J. Neurotoxicity of substituted amphetamines: molecular and cellular mechanisms. Neurotox Res. 2007;11:183–202.

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Eibl JK, Abdallah Z, Ross GM. Zinc-metallothionein: a potential mediator of antioxidant defence mechanisms in response to dopamine induced stress. Can J Physiol Pharm. 2010;88:305–12.

    CAS  Article  Google Scholar 

  21. 21.

    Cuajungco MP, Lees GJ. Zinc metabolism in the brain: relevance to human neurodegenerative disorders. Neurobiol Dis. 1997;4:137–69.

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Wigner P, Czarny P, Galecki P, Su KP, Sliwinski T. The molecular aspects of oxidative & nitrosative stress and the tryptophan catabolites pathway (TRYCATs) as potential causes of depression. Psychiatry Res. 2018;262:566–74.

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Hayashi Y, Majewska AK. Dendritic spine geometry: functional implication and regulation. Neuron. 2005;46:529–32.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Segal M. Dendritic spines, synaptic plasticity and neuronal survival: activity shapes dendritic spines to enhance neuronal viability. Eur J Neurosci. 2010;31:2178–84.

    PubMed  Article  Google Scholar 

  25. 25.

    Pérez-Rodríguez M, Arroyo-García LE, Prius-Mengual J, Andrade-Talavera Y, Armengol JA, Pérez-Villegas E, et al. Adenosine receptor-mediated developmental loss of spike timing-dependent depression in the hippocampus. Cereb Cortex. 2019;29:3266–3281.

    PubMed  Article  Google Scholar 

  26. 26.

    Fiala JC, Spacek J, Harris KM. Dendritic spine pathology: cause or consequence of neurological disorders? Brain Res Brain Res Rev. 2002;39:29–54.

    PubMed  Article  Google Scholar 

  27. 27.

    Blanpied TA, Ehlers MD. Microanatomy of dendritic spines: emerging principles of synaptic pathology in psychiatric and neurological disease. Biol Psychiatry. 2004;55:1121–7.

    PubMed  Article  Google Scholar 

  28. 28.

    Tendilla-Beltrán H, Antonio Vázquez-Roque R, Judith Vázquez-Hernández A, Garcés-Ramírez L, Flores G. Exploring the dendritic spine pathology in a schizophrenia-related neurodevelopmental animal model. Neuroscience. 2019;396:36–45.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Hernandez-Hernandez EM, Caporal Hernández K, Vázquez-Roque RA, Díaz A, de la Cruz F, Flores G. The neuropeptide-12 improves recognition memory and neural plasticity of the limbic system in old rats. Synapse. 2018;72:e22036.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Tendilla-Beltrán H, Meneses-Prado S, Vázquez-Roque RA, Tapia-Rodríguez M, Vázquez-Hernández AJ, Coatl-Cuaya H, et al. Risperidone ameliorates prefrontal cortex neural atrophy and oxidative/nitrosative stress in brain and peripheral blood of rats with neonatal ventral hippocampus lesion. J Neurosci. 2019;39:8584–99.

    PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Vázquez‐Roque RA, Ramos B, Tecuatl C, Juárez I, Adame A, de la Cruz, et al. Chronic administration of the neurotrophic agent cerebrolysin ameliorates the behavioral and morphological changes induced by neonatal ventral hippocampus lesion in a rat model of schizophrenia. J Neurosci Res. 2012;90:288–306.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Gibb R, Kolb B. A method for vibratome sectioning of Golgi-Cox stained whole rat brain. J Neurosci Methods. 1998;79:1–4.

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Paxinos G, Watson C. The rat brain in stereotaxic coordinates, 2nd edn. New York: Academic Press; 1986.

  34. 34.

    Kolb B, Forgie M, Gibb R, Gorny G, Rowntree S. Age, experience and the changing brain. Neurosci Biobehav Rev. 1998;22:143–59.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Sholl DA. Dendritic organization in the neurons of the visual and motor cortices of the cat. J Anat. 1953;87:387–406.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Flores G, Alquicer G, Silva-Gómez AB, Zaldivar G, Stewart J, Quirion R, et al. Alterations in dendritic morphology of prefrontal cortical and nucleus accumbens neurons in post-pubertal rats after neonatal excitotoxic lesions of the ventral hippocampus. Neuroscience. 2005;133:463–70.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Martínez-Tellez R, Gómez-Villalobos MJ, Flores G. Alteration in dendritic morphology of cortical neurons in rats with diabetes mellitus induced by streptozocin. Brain Res. 2005;1048:108–15.

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Silva-Gómez AB, Bermudez M, Quirion R, Srivastava LK, Picazo O, Flores G. Comparative behavioral changes between male and female postpubertal rats following neonatal excitotoxic lesions of the ventral hippocampus. Brain Res. 2003;973:285–92.

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Tendilla-Beltrán H, Arroyo-García LE, Díaz A, Camacho-Abrego I, de la Cruz F, Rodriguez-Moreno A, et al. The effects of amphetamine exposure on juvenile rats on the neuronal morphology of the limbic system at prepubertal, pubertal and postpubertal ages. J Chem Neuroanat. 2016;77:68–77.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Bello-Medina PC, Flores G, Quirarte GL, McGaugh JL, Prado-Alcalá RA. Mushroom spine dynamics in medium spiny neurons of dorsal striatum associated with memory of moderate and intense training. Proc Natl Acad Sci USA 2016;113:E6516–25.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Tellez-Merlo G, Morales-Medina JC, Camacho-Abrego I, Juaréz-Díaz I, Aguilar-Alonso P, de la Cruz F, et al. Prenatal immune challenge induces behavioral deficits, neuronal remodeling, and increases brain nitric oxide and zinc levels in the male rat offspring. Neuroscience. 2019;406:594–605.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Eaton DL, Toal BF. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol Appl Pharm. 1982;66:134–42.

    CAS  Article  Google Scholar 

  43. 43.

    Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351–8.

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Tindell AJ, Berridge KC, Jun Z, Peciña S, Aldridge JW. Ventral pallidal neurons code incentive motivation: amplification by mesolimbic sensitization and amphetamine. Eur J Neurosci. 2005;22:2617–34.

    PubMed  Article  Google Scholar 

  45. 45.

    Zhu J, Chen Y, Zhao N, Cao G, Dang Y, Chen T. Distinct roles of dopamine D3 receptors in modulating methamphetamine-induced behavioral sensitization and ultrastructural plasticity in the shell of the nucleus accumbens. J Neurosci Res. 2012;90:895–904.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Reske M, Eidt CA, Delis DC, Paulus MP. Nondependent stimulant users of cocaine and prescription amphetamines show verbal learning and memory deficits. Biol Psychiatry. 2010;68:762–9.

    PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Brown MW, Aggleton JP. Recognition memory: what are the roles of the perirhinal cortex and hippocampus? Nat Rev Neurosci. 2001;2:51–61.

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Bevins RA, Besheer J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ‘recognition memory’. Nat Protoc. 2006;1:1306–11.

    PubMed  Article  Google Scholar 

  49. 49.

    Kristiansen SL, Nyengaard JR. Digital stereology in neuropathology. APMIS. 2012;120:327–40.

    PubMed  Article  Google Scholar 

  50. 50.

    Kuczenski R, Everall IP, Crews L, Adame A, Grant I, Masliah E. Escalating dose-multiple binge methamphetamine exposure results in degeneration of the neocortex and limbic system in the rat. Exp Neurol 2007;207:42–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Crombag HS, Gorny G, Li Y, Kolb B, Robinson TE. Opposite effects of amphetamine self-administration experience on dendritic spines in the medial and orbital prefrontal cortex. Cereb Cortex. 2005;15:341–8.

    PubMed  Article  Google Scholar 

  52. 52.

    Singer BF, Tanabe LM, Gorny G, Jake-Matthews C, Li Y, Kolb B, et al. Amphetamine-induced changes in dendritic morphology in rat forebrain correspond to associative drug conditioning rather than nonassociative drug sensitization. Biol Psychiatry. 2009;65:835–40.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Regev L, Baram TZ. Corticotropin releasing factor in neuroplasticity. Front Neuroendocrinol. 2014;35:171–9.

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Mirzayans R, Andrais B, Kumar P, Murray D. The growing complexity of cancer cell response to DNA-damaging agents: caspase 3 mediates cell death or survival? Int J Mol Sci. 2016;17:708.

    PubMed Central  Article  CAS  Google Scholar 

  55. 55.

    Marreiro DD, Cruz KJ, Morais JB, Beserra JB, Severo JS, de Oliveira AR. Zinc and oxidative stress: current mechanisms. Antioxidants. 2017;6.

  56. 56.

    Travaglia A, La Mendola D, Magrì A, Pietropaolo A, Nicoletti VG, Grasso G, et al. Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives. Inorg Chem. 2013;52:11075–83.

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Szewczyk B. Zinc homeostasis and neurodegenerative disorders. Front Aging Neurosci. 2013;5:33.

  58. 58.

    Baltaci AK, Yuce K, Mogulkoc R. Zinc metabolism and metallothioneins. Biol Tra Ele Rese. 2017;55:223–33.

    CAS  Google Scholar 

  59. 59.

    Juárez-Rebollar D, Rios C, Nava-Ruíz C, Méndez-Armenta M. Metallothionein in brain disorders. Oxid Med Cell Longev. 2017;2017:5828056.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  60. 60.

    Vasto S, Mocchegiani E, Malavolta M, Cuppari I, Listi F, Nuzzo D, et al. Zinc and inflammatory/immune response in aging. Ann N Y Acad Sci. 2007;1100:111–22.

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Kang K, Lee SW, Han JE, Choi JW, Song MR. The complex morphology of reactive astrocytes controlled by fibroblast growth factor signaling. Glia. 2014;62:1328.

    PubMed  Article  Google Scholar 

  62. 62.

    Yang K, Broussard JI, Levine AT, Jenson D, Arenkiel BR, Dani JA. Dopamine receptor activity participates in hippocampal synaptic plasticity associated with novel object recognition. Eur J Neurosci. 2017;45:138–46.

    PubMed  Article  Google Scholar 

  63. 63.

    Sjulson L, Peyrache A, Cumpelik A, Cassataro D, Buzsáki G. Cocaine place conditioning strengthens location-specific hippocampal coupling to the nucleus accumbens. Neuron. 2018;98:926–34.e5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. 64.

    Wang X, Pal R, Chen XW, Limpeanchob N, Kumar KN, Michaelis EK. High intrinsic oxidative stress may underlie selective vulnerability of the hippocampal CA1 region. Brain Res. 2005;140:120–6.

    CAS  Google Scholar 

  65. 65.

    Wilde GJ, Pringle AK, Wright P, Iannotti F. Differential vulnerability of the CA1 and CA3 subfields of the hippocampus to superoxide and hydroxyl radicals in vitro. J Neurochem. 1997;69:883–6.

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Cadet JL, Patel R, Jayanthi S. Compulsive methamphetamine taking and abstinence in the presence of adverse consequences: epigenetic and transcriptional consequences in the rat brain. Pharm Biochem Behav. 2019;179:98–108.

    CAS  Article  Google Scholar 

Download references


LEA-G and HT-B acknowledge CONACYT for the fellowship. EB, PA-A, AD, RAV-R, FDLC, EM, and GF acknowledge the “Sistema Nacional de Investigadores” of Mexico for memberships. Thanks to Miguel Tapia-Rodríguez (Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México) for stereological procedures assistance and to Professor Robert Simpson for editing the English language text.


Funding for this study was provided by grants from the PRODEP (CA-BUAP-120) and the CONACYT grant (No. 252808) to GF and MINECO/FEDER (BFU2012-38208) and the Junta de Andalucía (P11-CVI-7290) to AR-M. None of the funding institutions had any further role in the study design, the collection or interpretation of data, analyses, the writing of the report or the decision to submit the paper for publication.

Author information




LEA-G, HT-B, AR-M, RAV-R, FDLC, and GF designed the study and wrote the protocol. LEA-G, HT-B, EEJT, AD, PA-A, EB, and EM performed the experiments. LEA-G, AR-M, and GF performed the literature searches and analysis and LEA-G and GF undertook the statistical analysis. LEA-G, AR-M, and GF wrote the first draft of the manuscript. All contributing authors have approved the final manuscript.

Corresponding author

Correspondence to Gonzalo Flores.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Arroyo-García, L.E., Tendilla-Beltrán, H., Vázquez-Roque, R.A. et al. Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors. Mol Psychiatry (2020).

Download citation

Further reading