Advertisement

pp 1-26 | Cite as

In Vitro and In Vitro Models for Alcohol Toxicity to the Extracellular Matrix of Astrocytes

  • Xiaolu Zhang
  • Marina Guizzetti
Protocol
Part of the Methods in Pharmacology and Toxicology book series

Abstract

Ethanol exposure exerts profound effects on the developing brain, which lead to cognitive impairment and behavioral functional disorders as seen in fetal alcohol spectrum disorders (FASD). Astrocytes play a major role in neuritogenesis and neurite outgrowth by modulating neuronal plasticity through the regulation of the composition of the extracellular matrix (ECM), which is involved in the extension of neurites and the formation and remodeling of synaptic connections. Ethanol affects the levels of neuritogenic ECM proteins release by astrocytes and inhibits neuronal plasticity. In this unit, we describe in vitro and in vivo models of developmental ethanol exposure in which to investigate mechanisms of ethanol-induced alterations in the astrocyte ECM leading to abnormal neuronal development. The described in vitro model consists of primary astrocyte cultures exposed to ethanol in sealed chambers and of astrocyte/neuron co-cultures in which neurons are in contact with astrocytes previously exposed to ethanol. The in vivo model consists of neonatal rat pups exposed to ethanol via intragastric intubation. The developmental stage of the brain of neonatal rats in this model is comparable to the developmental stage of the human brain during the third trimester of gestation. This in vivo FASD model recapitulates some of the behavioral effects of developmental ethanol exposure in humans. A description of how to determine changes in ECM proteins after in vitro and in vivo ethanol exposure will also be provided, as well as procedures for assessing neurite outgrowth in the in vitro model and dendrite arborization in the in vivo model. These two models are instrumental to the investigation of how ethanol, during brain development, alters the composition of astrocyte-secreted ECM leading to altered brain development.

Keywords

Alcohol toxicity Astrocytes ECM In vitro In vivo 

Notes

Acknowledgments

This work was supported by VA Merit Review Award # I01BX001819 and by NIH/NIAAA R01AA021468, R21AA021876, and R01AA022948 and by facilities and resources at the VA Portland Health Care System. The opinions expressed in this paper are solely those of the authors. The contents do not represent the views of the United States (US) Department of Veterans Affairs or the US government.

References

  1. 1.
    Gerace E, Landucci E, Totti A, Bani D, Guasti D, Baronti R, Moroni F, Mannaioni G, Pellegrini-Giampietro DE (2016) Ethanol toxicity during brain development: alterations of excitatory synaptic transmission in immature organotypic hippocampal slice cultures. Alcohol Clin Exp Res 40:706–716Google Scholar
  2. 2.
    Chedotal A, Richards LJ (2010) Wiring the brain: the biology of neuronal guidance. Cold Spring Harb Perspect Biol 2:a001917Google Scholar
  3. 3.
    Higgins D, Burack M, Lein P, Banker G (1997) Mechanisms of neuronal polarity. Curr Opin Neurobiol 7:599–604Google Scholar
  4. 4.
    Marin O, Rubenstein JL (2003) Cell migration in the forebrain. Annu Rev Neurosci 26:441–483Google Scholar
  5. 5.
    Ullian EM, Sapperstein SK, Christopherson KS, Barres BA (2001) Control of synapse number by glia. Science 291:657–661Google Scholar
  6. 6.
    Garcia O, Torres M, Helguera P, Coskun P, Busciglio J (2010) A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down’s syndrome. PLoS One 5:e14200Google Scholar
  7. 7.
    Jacobs S, Doering LC (2010) Astrocytes prevent abnormal neuronal development in the fragile x mouse. J Neurosci 30:4508–4514Google Scholar
  8. 8.
    Muller HW, Junghans U, Kappler J (1995) Astroglial neurotrophic and neurite-promoting factors. Pharmacol Ther 65:1–18Google Scholar
  9. 9.
    Martinez R, Gomes FC (2002) Neuritogenesis induced by thyroid hormone-treated astrocytes is mediated by epidermal growth factor/mitogen-activated protein kinase-phosphatidylinositol 3-kinase pathways and involves modulation of extracellular matrix proteins. J Biol Chem 277:49311–49318Google Scholar
  10. 10.
    Tom VJ, Doller CM, Malouf AT, Silver J (2004) Astrocyte-associated fibronectin is critical for axonal regeneration in adult white matter. J Neurosci 24:9282–9290Google Scholar
  11. 11.
    Askari JA, Buckley PA, Mould AP, Humphries MJ (2009) Linking integrin conformation to function. J Cell Sci 122:165–170Google Scholar
  12. 12.
    Johnson KM, Milner R, Crocker SJ (2015) Extracellular matrix composition determines astrocyte responses to mechanical and inflammatory stimuli. Neurosci Lett 600:104–109Google Scholar
  13. 13.
    Fernandez-Monreal M, Lopez-Atalaya JP, Benchenane K, Leveille F, Cacquevel M, Plawinski L, MacKenzie ET, Bu G, Buisson A, Vivien D (2004) Is tissue-type plasminogen activator a neuromodulator? Mol Cell Neurosci 25:594–601Google Scholar
  14. 14.
    Frischknecht R, Chang KJ, Rasband MN, Seidenbecher CI (2014) Neural ECM molecules in axonal and synaptic homeostatic plasticity. Prog Brain Res 214:81–100Google Scholar
  15. 15.
    Saito M, Chakraborty G, Hui M, Masiello K (2016) Ethanol-induced neurodegeneration and glial activation in the developing brain. Brain Sci 6.  https://doi.org/10.3390/brainsci6030031
  16. 16.
    Sofroniew MV (2014) Astrogliosis. Cold Spring Harb Perspect Biol 7:a020420Google Scholar
  17. 17.
    Guizzetti M, Zhang X, Goeke C, Gavin DP (2014) Glia and neurodevelopment: focus on fetal alcohol Spectrum disorders. Front Pediatr 2:123Google Scholar
  18. 18.
    Wilhelm CJ, Guizzetti M (2016) Fetal alcohol spectrum disorders: an overview from the glia perspective. Front Integr Neurosci 9:65Google Scholar
  19. 19.
    Ren LQ, Garrett DK, Syapin M, Syapin PJ (2000) Differential fibronectin expression in activated C6 glial cells treated with ethanol. Mol Pharmacol 58:1303–1309Google Scholar
  20. 20.
    Trindade P, Hampton B, Manhaes AC, Medina AE (2016) Developmental alcohol exposure leads to a persistent change on astrocyte secretome. J Neurochem 137:730–743Google Scholar
  21. 21.
    Guizzetti M, Moore NH, Giordano G, VanDeMark KL, Costa LG (2010) Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors. Glia 58:1395–1406Google Scholar
  22. 22.
    Moore NH, Costa LG, Shaffer SA, Goodlett DR, Guizzetti M (2009) Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation. J Neurochem 108:891–908Google Scholar
  23. 23.
    Zhang X, Kusumo H, Sakharkar AJ, Pandey SC, Guizzetti M (2014) Regulation of DNA methylation by ethanol induces tissue plasminogen activator expression in astrocytes. J Neurochem 128:344–349Google Scholar
  24. 24.
    Calabrese EJ (2008) Enhancing and regulating neurite outgrowth. Crit Rev Toxicol 38:391–418Google Scholar
  25. 25.
    Johnson TB, Goodlett CR (2002) Selective and enduring deficits in spatial learning after limited neonatal binge alcohol exposure in male rats. Alcohol Clin Exp Res 26:83–93Google Scholar
  26. 26.
    Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. J Neurosci 8:1454–1468Google Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Northern Jiangsu People’s HospitalYangzhouChina
  2. 2.Department of Behavioral NeuroscienceOregon Health and Science University, VA Portland Health Care SystemPortlandUSA

Personalised recommendations