Ethanol Exposure Transiently Elevates but Persistently Inhibits Tyrosine Kinase Activity and Impairs the Growth of the Nascent Apical Dendrite

  • Dandan Wang
  • Joshua Enck
  • Brian W. Howell
  • Eric C. OlsonEmail author


Dendritogenesis can be impaired by exposure to alcohol, and aspects of this impairment share phenotypic similarities to dendritic defects observed after blockade of the Reelin-Dab1 tyrosine kinase signaling pathway. In this study, we find that 10 min of alcohol exposure (400 mg/dL ethanol) by itself causes an unexpected increase in tyrosine phosphorylation of many proteins including Src and Dab1 that are essential downstream effectors of Reelin signaling. This increase in phosphotyrosine is dose-dependent and blockable by selective inhibitors of Src Family Kinases (SFKs). However, the response is transient, and phosphotyrosine levels return to baseline after 30 min of continuous ethanol exposure, both in vitro and in vivo. During this latter period, Src is inactivated and Reelin application cannot stimulate Dab1 phosphorylation. This suggests that ethanol initially activates but then silences the Reelin-Dab1 signaling pathway by brief activation and then sustained inactivation of SFKs. Time-lapse analyses of dendritic growth dynamics show an overall decrease in growth and branching compared to controls after ethanol-exposure that is similar to that observed with Reelin-deficiency. However, unlike Reelin-signaling disruptions, the dendritic filopodial speeds are decreased after ethanol exposure, and this decrease is associated with sustained dephosphorylation and activation of cofilin, an F-actin severing protein. These findings suggest that persistent Src inactivation coupled to cofilin activation may contribute to the dendritic disruptions observed with fetal alcohol exposure.


Fetal alcohol syndrome disorder Cortical development Src kinases Dab1 Dendritogenesis Cofilin 



fetal alcohol spectrum disorder


Src family kinases




phenylarsine oxide




Reelin-conditioned medium



We thank members of the Olson lab and Krysten O’Hara for assistance and valuable input on the project.

Author Contribution

EO and DW designed the study with significant input from BH. DW and JE performed the study. EO and DW wrote the manuscript with comments and edits from BH, JE, and members of the Developmental Exposure to Alcohol Research Center (DEARC).

Funding Information

This work was supported by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) via the Developmental Exposure to Alcohol Research Center (DEARC) P50AA017823-10.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12035_2019_1473_Fig8_ESM.png (360 kb)
Supplementary Figure 1

a and b, Exposure to increasing concentrations (0-0.75%) of EtOH did not reduce cell viability of embryonic cortical neurons as determined by Calcein AM / Propidium Iodide (PI) assay. Live cells internalize the Calcein dye but are not stained with propidium iodide. Cortical cultures were treated with different concentration of EtOH (0.25%, 0.5% and 0.75%) for different time (1h, 4h or 16h) followed with 30 min incubation of 3 μM Calcein AM and 5 μM PI. Wells containing cells treated with 0.2% Triton X-100 (15min) was used as control. Fluorescence signal was detected at 485nm and 528nm. Data was normalized to untreated cells. c, Membrane cholesterol manipulation did not significantly alter EtOH-induced tyrosine phosphorylation. Cortical cultures were treated with 10 min of 0.5% EtOH after cholesterol supplementation (500μM, 30 min) or cholesterol depletion using methyl-β-cyclodextrin (MβCD, 500μM, 30 min). PY99 expression was determined by western blotting. GAPDH was used as loading control. d, Quantification of blots from figure c. One-way ANOVA followed by Bonferroni post hoc test was used between different groups. NS, p > 0.05, # p < 0.001. (PNG 360 kb)

12035_2019_1473_MOESM1_ESM.tif (2.9 mb)
High Resolution Image (TIF 2930 kb)


  1. 1.
    May PA, Chambers CD, Kalberg WO, Zellner J, Feldman H, Buckley D, Kopald D, Hasken JM et al (2018) Prevalence of Fetal Alcohol Spectrum Disorders in 4 US Communities. JAMA 319:474–482CrossRefGoogle Scholar
  2. 2.
    Kodituwakku PW, Handmaker NS, Cutler SK, Weathersby EK, Handmaker SD (1995) Specific impairments in self-regulation in children exposed to alcohol prenatally. Alcohol Clin Exp Res 19:1558–1564CrossRefGoogle Scholar
  3. 3.
    Mattson SN, Riley EP (1998) A review of the neurobehavioral deficits in children with fetal alcohol syndrome or prenatal exposure to alcohol. Alcohol Clin Exp Res 22:279–294CrossRefGoogle Scholar
  4. 4.
    Rodriguez CI, Davies S, Calhoun V, Savage DD, Hamilton DA (2016) Moderate prenatal alcohol exposure alters functional connectivity in the adult rat brain. Alcohol Clin Exp Res 40:2134–2146CrossRefGoogle Scholar
  5. 5.
    Ishii S, Hashimoto-Torii K (2015) Impact of prenatal environmental stress on cortical development. Front Cell Neurosci 9:207CrossRefGoogle Scholar
  6. 6.
    Yanni PA, Lindsley TA (2000) Ethanol inhibits development of dendrites and synapses in rat hippocampal pyramidal neuron cultures. Brain Res Dev Brain Res 120:233–243CrossRefGoogle Scholar
  7. 7.
    Lindsley TA, Mazurkiewicz JE (2013) Ethanol modulates spontaneous calcium waves in axonal growth cones in vitro. Brain Sci 3:615–626CrossRefGoogle Scholar
  8. 8.
    Servais L, Hourez R, Bearzatto B, Gall D, Schiffmann SN, Cheron G (2007) Purkinje cell dysfunction and alteration of long-term synaptic plasticity in fetal alcohol syndrome. Proc Natl Acad Sci U S A 104:9858–9863CrossRefGoogle Scholar
  9. 9.
    O'Dell RS, Cameron DA, Zipfel WR, Olson EC (2015) Reelin prevents apical neurite retraction during terminal translocation and dendrite initiation. J Neurosci 35:10659–10674CrossRefGoogle Scholar
  10. 10.
    Picken Bahrey HL, Moody WJ (2003) Early development of voltage-gated ion currents and firing properties in neurons of the mouse cerebral cortex. J Neurophysiol 89:1761–1773CrossRefGoogle Scholar
  11. 11.
    Cameron DA, Middleton FA, Chenn A, Olson EC (2012) Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development. BMC Neurosci 13:90CrossRefGoogle Scholar
  12. 12.
    Guadagnoli T, Caltana L, Vacotto M, Gironacci MM, Brusco A (2016) Direct effects of ethanol on neuronal differentiation: an in vitro analysis of viability and morphology. Brain Res Bull 127:177–186CrossRefGoogle Scholar
  13. 13.
    Goeke CM, Roberts ML, Hashimoto JG, Finn DA, Guizzetti M (2018) Neonatal ethanol and choline treatments alter the morphology of developing rat hippocampal pyramidal neurons in opposite directions. Neuroscience 374:13–24CrossRefGoogle Scholar
  14. 14.
    Fontaine CJ, Patten AR, Sickmann HM, Helfer JL, Christie BR (2016) Effects of pre-natal alcohol exposure on hippocampal synaptic plasticity: sex, age and methodological considerations. Neurosci Biobehav Rev 64:12–34CrossRefGoogle Scholar
  15. 15.
    Louth EL, Luctkar HD, Heney KA, Bailey CDC (2018) Developmental ethanol exposure alters the morphology of mouse prefrontal neurons in a layer-specific manner. Brain Res 1678:94–105CrossRefGoogle Scholar
  16. 16.
    Powrozek TA, Olson EC (2012) Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons. Alcohol 46:619–627CrossRefGoogle Scholar
  17. 17.
    O'Dell RS, Ustine CJ, Cameron DA, Lawless SM, Williams RM, Zipfel WR, Olson EC (2012) Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone. Neural Dev 7:25CrossRefGoogle Scholar
  18. 18.
    Jossin Y, Cooper JA (2011) Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex. Nat Neurosci 14:697–703CrossRefGoogle Scholar
  19. 19.
    Jossin Y, Goffinet AM (2007) Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth. Mol Cell Biol 27:7113–7124CrossRefGoogle Scholar
  20. 20.
    Kuo G, Arnaud L, Kronstad-O'Brien P, Cooper JA (2005) Absence of Fyn and Src causes a reeler-like phenotype. J Neurosci 25:8578–8586CrossRefGoogle Scholar
  21. 21.
    La Torre A, del Mar Masdeu M, Cotrufo T, Moubarak RS, del Rio JA, Comella JX, Soriano E, Urena JM (2013) A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching. Cell Death Dis 4:e602CrossRefGoogle Scholar
  22. 22.
    Finsterwald C, Fiumelli H, Cardinaux JR, Martin JL (2010) Regulation of dendritic development by BDNF requires activation of CRTC1 by glutamate. J Biol Chem 285:28587–28595CrossRefGoogle Scholar
  23. 23.
    Qiu S, Lu Z, Levitt P (2014) MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus. J Neurosci 34:16166–16179CrossRefGoogle Scholar
  24. 24.
    Oliva C, Hassan BA (2017) Receptor tyrosine kinases and phosphatases in neuronal wiring: insights from drosophila. Curr Top Dev Biol 123:399–432CrossRefGoogle Scholar
  25. 25.
    D’Arcangelo G, Miao GG, Chen SC, Soares HD, Morgan JI, Curran T (1995) A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374:719–723CrossRefGoogle Scholar
  26. 26.
    Pinto-Lord MC, Evrard P, Caviness VS Jr (1982) Obstructed neuronal migration along radial glial fibers in the neocortex of the reeler mouse: a Golgi-EM analysis. Brain Res 256:379–393CrossRefGoogle Scholar
  27. 27.
    Olson EC, Kim S, Walsh CA (2006) Impaired neuronal positioning and dendritogenesis in the neocortex after cell-autonomous Dab1 suppression. J Neurosci 26:1767–1775CrossRefGoogle Scholar
  28. 28.
    Bock HH, Herz J (2003) Reelin activates SRC family tyrosine kinases in neurons. Curr Biol 13:18–26CrossRefGoogle Scholar
  29. 29.
    Howell BW, Herrick TM, Hildebrand JD, Zhang Y, Cooper JA (2000) Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development. Curr Biol 10:877–885CrossRefGoogle Scholar
  30. 30.
    Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 271:695–701CrossRefGoogle Scholar
  31. 31.
    Oetken C, von Willebrand M, Marie-Cardine A, Pessa-Morikawa T, Stahls A, Fisher S, Mustelin T (1994) Induction of hyperphosphorylation and activation of the p56lck protein tyrosine kinase by phenylarsine oxide, a phosphotyrosine phosphatase inhibitor. Mol Immunol 31:1295–1302CrossRefGoogle Scholar
  32. 32.
    Wing DR, Harvey DJ, Hughes J, Dunbar PG, McPherson KA, Paton WD (1982) Effects of chronic ethanol administration on the composition of membrane lipids in the mouse. Biochem Pharmacol 31:3431–3439CrossRefGoogle Scholar
  33. 33.
    Mrak RE (1992) Opposite effects of dimethyl sulfoxide and ethanol on synaptic membrane fluidity. Alcohol 9:513–517CrossRefGoogle Scholar
  34. 34.
    Laura Toppozini CLA, Barrett MA, Zheng S, Luo L, Nanda H, Sakai VG, Rheinstädter MC (2012) Partitioning of ethanol into lipid membranes and its effect on fluidity and permeability as seen by X-ray and neutron scattering. Soft Matter 8Google Scholar
  35. 35.
    Sefton BM, Trowbridge IS, Cooper JA, Scolnick EM (1982) The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid. Cell 31:465–474CrossRefGoogle Scholar
  36. 36.
    Liang X, Nazarian A, Erdjument-Bromage H, Bornmann W, Tempst P, Resh MD (2001) Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction. J Biol Chem 276:30987–30994CrossRefGoogle Scholar
  37. 37.
    Chai X, Forster E, Zhao S, Bock HH, Frotscher M (2009) Reelin stabilizes the actin cytoskeleton of neuronal processes by inducing n-cofilin phosphorylation at serine3. J Neurosci 29:288–299CrossRefGoogle Scholar
  38. 38.
    Flynn KC, Hellal F, Neukirchen D, Jacob S, Tahirovic S, Dupraz S, Stern S, Garvalov BK et al (2012) ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain. Neuron 76:1091–1107CrossRefGoogle Scholar
  39. 39.
    Liu SL, May JR, Helgeson LA, Nolen BJ (2013) Insertions within the actin core of actin-related protein 3 (Arp3) modulate branching nucleation by Arp2/3 complex. J Biol Chem 288:487–497CrossRefGoogle Scholar
  40. 40.
    Mullins RD, Heuser JA, Pollard TD (1998) The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci U S A 95:6181–6186CrossRefGoogle Scholar
  41. 41.
    Piper M, Lee AC, van Horck FP, McNeilly H, Lu TB, Harris WA, Holt CE (2015) Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones. Neural Dev 10:3CrossRefGoogle Scholar
  42. 42.
    Tian D, Diao M, Jiang Y, Sun L, Zhang Y, Chen Z, Huang S, Ou G (2015) Anillin regulates neuronal migration and neurite growth by linking RhoG to the actin cytoskeleton. Curr Biol 25:1135–1145CrossRefGoogle Scholar
  43. 43.
    Ohashi K (2015) Roles of cofilin in development and its mechanisms of regulation. Develop Growth Differ 57:275–290CrossRefGoogle Scholar
  44. 44.
    Rosario M, Schuster S, Juttner R, Parthasarathy S, Tarabykin V, Birchmeier W (2012) Neocortical dendritic complexity is controlled during development by NOMA-GAP-dependent inhibition of Cdc42 and activation of cofilin. Genes Dev 26:1743–1757CrossRefGoogle Scholar
  45. 45.
    Moriyama K, Iida K, Yahara I (1996) Phosphorylation of Ser-3 of cofilin regulates its essential function on actin. Genes Cells 1:73–86CrossRefGoogle Scholar
  46. 46.
    Nichols AJ, O'Dell RS, Powrozek TA, Olson EC Ex utero electroporation and whole hemisphere explants: a simple experimental method for studies of early cortical development. J Vis Exp.
  47. 47.
    Nadarajah B, Parnavelas JG (2002) Modes of neuronal migration in the developing cerebral cortex. Nat Rev Neurosci 3:423–432CrossRefGoogle Scholar
  48. 48.
    Chai X, Fan L, Shao H, Lu X, Zhang W, Li J, Wang J, Chen S et al (2015) Reelin induces branching of neurons and radial glial cells during corticogenesis. Cereb Cortex 25:3640–3653CrossRefGoogle Scholar
  49. 49.
    Cooper JA (2014) Molecules and mechanisms that regulate multipolar migration in the intermediate zone. Front Cell Neurosci 8:386PubMedPubMedCentralGoogle Scholar
  50. 50.
    Rashid M, Belmont J, Carpenter D, Turner CE, Olson EC (2017) Neural-specific deletion of the focal adhesion adaptor protein paxillin slows migration speed and delays cortical layer formation. Development 144:4002–4014CrossRefGoogle Scholar
  51. 51.
    Xu J, Kurup P, Foscue E, Lombroso PJ (2015) Striatal-enriched protein tyrosine phosphatase regulates the PTPalpha/Fyn signaling pathway. J Neurochem 134:629–641CrossRefGoogle Scholar
  52. 52.
    Yokoyama N, Miller WT (2003) Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck. J Biol Chem 278:47713–47723CrossRefGoogle Scholar
  53. 53.
    Ikeda K, Wang LH, Torres R, Zhao H, Olaso E, Eng FJ, Labrador P, Klein R et al (2002) Discoidin domain receptor 2 interacts with Src and Shc following its activation by type I collagen. J Biol Chem 277:19206–19212CrossRefGoogle Scholar
  54. 54.
    Chai X, Zhao S, Fan L, Zhang W, Lu X, Shao H, Wang S, Song L et al (2016) Reelin and cofilin cooperate during the migration of cortical neurons: a quantitative morphological analysis. Development 143:1029–1040CrossRefGoogle Scholar
  55. 55.
    Jossin Y, Ogawa M, Metin C, Tissir F, Goffinet AM (2003) Inhibition of SRC family kinases and non-classical protein kinases C induce a reeler-like malformation of cortical plate development. J Neurosci 23:9953–9959CrossRefGoogle Scholar
  56. 56.
    Yip YP, Kronstadt-O'Brien P, Capriotti C, Cooper JA, Yip JW (2007) Migration of sympathetic preganglionic neurons in the spinal cord is regulated by Reelin-dependent Dab1 tyrosine phosphorylation and CrkL. J Comp Neurol 502:635–643CrossRefGoogle Scholar
  57. 57.
    Howell BW, Gertler FB, Cooper JA (1997) Mouse disabled (mDab1): a Src binding protein implicated in neuronal development. EMBO J 16:121–132CrossRefGoogle Scholar
  58. 58.
    Feng L, Allen NS, Simo S, Cooper JA (2007) Cullin 5 regulates Dab1 protein levels and neuron positioning during cortical development. Genes Dev 21:2717–2730CrossRefGoogle Scholar
  59. 59.
    de Vivo L, Landi S, Panniello M, Baroncelli L, Chierzi S, Mariotti L, Spolidoro M, Pizzorusso T et al (2013) Extracellular matrix inhibits structural and functional plasticity of dendritic spines in the adult visual cortex. Nat Commun 4:1484CrossRefGoogle Scholar
  60. 60.
    Novak U, Kaye AH (2000) Extracellular matrix and the brain: components and function. J Clin Neurosci 7:280–290CrossRefGoogle Scholar
  61. 61.
    Popp RL, Dertien JS (2008) Actin depolymerization contributes to ethanol inhibition of NMDA receptors in primary cultured cerebellar granule cells. Alcohol 42:525–539CrossRefGoogle Scholar
  62. 62.
    Romero AM, Esteban-Pretel G, Marin MP, Ponsoda X, Ballestin R, Canales JJ, Renau-Piqueras J (2010) Chronic ethanol exposure alters the levels, assembly, and cellular organization of the actin cytoskeleton and microtubules in hippocampal neurons in primary culture. Toxicol Sci 118:602–612CrossRefGoogle Scholar
  63. 63.
    Romero AM, Renau-Piqueras J, Pilar Marin M, Timoneda J, Berciano MT, Lafarga M, Esteban-Pretel G (2013) Chronic alcohol alters dendritic spine development in neurons in primary culture. Neurotox Res 24:532–548CrossRefGoogle Scholar
  64. 64.
    dos Remedios CG, Chhabra D, Kekic M, Dedova IV, Tsubakihara M, Berry DA, Nosworthy NJ (2003) Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev 83:433–473CrossRefGoogle Scholar
  65. 65.
    Wang X, Qiu R, Tsark W, Lu Q (2007) Rapid promoter analysis in developing mouse brain and genetic labeling of young neurons by doublecortin-DsRed-express. J Neurosci Res 85:3567–3573CrossRefGoogle Scholar
  66. 66.
    Nichols AJ, Olson EC (2010) Reelin promotes neuronal orientation and dendritogenesis during preplate splitting. Cereb Cortex 20:2213–2223CrossRefGoogle Scholar
  67. 67.
    O'Dell R, Ustine CM, Lawless SM, Cameron DA, Williams CM, Zipfel WR, Olson EC (2011) Dendritogenesis and orientation of Layer 6 cortical neurons during early cortical development. Society for Neuroscience, Washington D.C.Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Neuroscience and PhysiologySUNY Upstate Medical UniversitySyracuseUSA
  2. 2.Developmental Exposure to Alcohol Research Center (DEARC)Binghamton UniversityBinghamtonUSA

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