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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Published: April 3rd, 2013

DOI:

10.3791/50271

1Department of Neuroscience and Physiology, SUNY Upstate Medical University
* These authors contributed equally

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.

The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

The mammalian cerebral cortex forms through the concerted proliferation, migration and differentiation of successively generated neurons. Each neuron is born in the ventricular zone (VZ) and migrates from the VZ into the intermediate zone (IZ), forming the cortical plate (CP) 1. As they pass through different cortical domains, the migrating neurons display multiple modes of migration 2,3 that depend on the extracellular environment and other cellular elements (e.g. radial glia) within the developing tissue. Cortical neurons then arrest migration at the top of the forming cortical plate in the coincident processes of neuronal migration ar....

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1. Ex utero Electroporations with Green Fluorescent Protein Expression Plasmid

  1. Plasmid DNA injection solutions are prepared with CAG-eGFP DNA 27 diluted to the final working concentration of 0.33 mg/ml in ddH2O. Qiagen Endo-Free Maxi-Preps are used to purify the plasmid from transformed bacteria. Fast green dye at ~0.02% (w/v final) is added to the DNA solution as an injection tracer.
  2. To prepare the surgical area, spray down bench top and dissecting microscope stage .......

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The embryonic rodent cortex exhibits a transverse neurogenetic gradient throughout the developmental period, such that lateral neocortex is approximately 1 day more mature than dorsal medial neocortex 28. The bulk of layer 6 neurons are thus generated (i.e. exhibit their final S-phase) on E12 in lateral cortex (also called Field 405) and at E13 in the dorsal medial cortex (also called Field 15). Preplate splitting begins approximately 1 day after Layer 6 neuron generation and thu.......

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We have improved and evaluated an experimental model - whole hemisphere explants - for the study of early cortical development (E13-E15). The model has proven useful for the analysis of migration and differentiation of the excitatory neuron lineage that constitutes layer 6 of the cerebral cortex 25,37 . The principle advantages of the system are 1) organotypic growth for 2 DIV, 2) simplicity of preparation, and 3) experimental access to the neurons for electroporation, pharmacological manipulation and imaging .......

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This work was supported grants from NINDS (NS066071) and the NIAAA. (P50AA017823) to ECO. The authors thank Dr. Robert Quinn and the staff in the Department of Laboratory Animal Resources for animal care. We thank Judson Belmont for technical support, Nicole Belletier for assistance as a Summer Undergraduate Research Fellow (SURF). We also thank Dr. David Cameron for comments and edits on an earlier version of the manuscript.

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Name Company Catalog Number Comments
Name Company Catalog Number Comments (optional)
Reagents      
DMEM/F12 + GlutaMAX GIBCO 10565  
G5 Supplement 100X Invitrogen 17503-012  
B27 Serum-Free Suppl. 50X Invitrogen 1504-044  
Pen / Strep Liquid 100X Invitrogen 15140-122  
HBSS 500 ml GIBCO 14025  
Culture insert collagen coated Costar 3492  
Bovine skin gelatin Sigma G9382  
Hoechst 33342 Invitrogen H1399  
Bovine Serum Albumin Sigma 7906  
EndoFree Plasmid Maxi Kit Qiagen 12362  
Equipment      
BTX 830 Electroporator Harvard Apparatus 450052  
Tweezer electrodes 10mm Harvard Apparatus 450166  
Incubator Billups Rothenberg MIC-101  
Hamilton syringe (5 uL) Hamilton 87930  
Hamilton syringe needle Hamilton 7803-04 Specify 1" and style 4
Dumont #5 Forceps FST 11251-10  
Fine Scissors Tough Cut 9 cm FST 14058-09  

  1. Rakic, P. Principles of neural cell migration. Experientia. 46, 882-891 (1990).
  2. Tabata, H., Nakajima, K. Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex. J. Neurosci. 23, 9996-10001 (2003).
  3. Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., Kriegstein, A. R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat. Neurosci. , (2004).
  4. Olson, E. C., Kim, S., Walsh, C. A. Impaired neuronal positioning and dendritogenesis in the neocortex after cell-autonomous Dab1 suppression. J. Neurosci. 26, 1767-1775 (2006).
  5. Takahashi, T., Goto, T., Miyama, S., Nowakowski, R. S., Caviness, V. S. Sequence of neuron origin and neocortical laminar fate: relation to cell cycle of origin in the developing murine cerebral wall. J. Neurosci. 19, 10357-10371 (1999).
  6. Marin-Padilla, M. Early prenatal ontogenesis of the cerebral cortex (neocortex) of the cat (Felis domestica). A Golgi study. I. The primordial neocortical organization. Z. Anat. Entwicklungsgesch. 134, 117-145 (1971).
  7. Nichols, A. J., Olson, E. C. Reelin promotes neuronal orientation and dendritogenesis during preplate splitting. Cerebral Cortex. 20, 2213-2223 (2010).
  8. Sheppard, A. M., Pearlman, A. L. Abnormal reorganization of preplate neurons and their associated extracellular matrix: an early manifestation of altered neocortical development in the reeler mutant mouse. J. Comp. Neurol. 378, 173-179 (1997).
  9. Manzini, M. C., Walsh, C. A. What disorders of cortical development tell us about the cortex: one plus one does not always make two. Current Opinion in Genetics & Development. 21, 333-339 (2011).
  10. Jones, L., Fischer, I., Levitt, P. Nonuniform alteration of dendritic development in the cerebral cortex following prenatal cocaine exposure. Cereb. Cortex. 6, 431-445 (1996).
  11. Olney, J. W. Fetal alcohol syndrome at the cellular level. Addict. Biol. 9, 137-149 (2004).
  12. Levitt, P., Reinoso, B., Jones, L. The critical impact of early cellular environment on neuronal development. Prev. Med. 27, 180-183 (1998).
  13. Stoppini, L., Buchs, P. A., Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods. 37, 173-182 (1991).
  14. O'Rourke, N. A., Dailey, M. E., Smith, S. J., McConnell, S. K. Diverse migratory pathways in the developing cerebral cortex. Science. 258, 299-302 (1992).
  15. Elias, L. A., Wang, D. D., Kriegstein, A. R. Gap junction adhesion is necessary for radial migration in the neocortex. Nature. 448, 901-907 (2007).
  16. Franco, S. J., Martinez-Garay, I., Gil-Sanz, C., Harkins-Perry, S. R., Muller, U. Reelin regulates cadherin function via Dab1/Rap1 to control neuronal migration and lamination in the neocortex. Neuron. 69, 482-497 (2011).
  17. Gotz, M., Bolz, J. Formation and preservation of cortical layers in slice cultures. J. Neurobiol. 23, 783-802 (1992).
  18. Cameron, R. S., Rakic, P. Identification of membrane proteins that comprise the plasmalemmal junction between migrating neurons and radial glial cells. J Neurosci. 14, 3139-3155 (1994).
  19. Lizarraga, S. B., Coser, K. R., Sabbagh, M., Morrow, E. M. Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex. J. Vis. Exp. (63), e3621 (2012).
  20. Jossin, Y., Goffinet, A. M. Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth. Mol. Cell Biol. 27, 7113-7124 (2007).
  21. Jossin, Y., Ogawa, M., Metin, C., Tissir, F., Goffinet, A. M. Inhibition of SRC family kinases and non-classical protein kinases C induce a reeler-like malformation of cortical plate development. J. Neurosci. 23, 9953-9959 (2003).
  22. Kingsbury, M. A., Rehen, S. K., Contos, J. J., Higgins, C. M., Chun, J. Non-proliferative effects of lysophosphatidic acid enhance cortical growth and folding. Nat. Neurosci. 6, 1292-1299 (2003).
  23. Rehen, S. K., et al. A new method of embryonic culture for assessing global changes in brain organization. J. Neurosci. Methods. 158, 100-108 (2006).
  24. Jossin, Y., et al. The central fragment of Reelin, generated by proteolytic processing in vivo, is critical to its function during cortical plate development. J. Neurosci. 24, 514-521 (2004).
  25. O'Dell, R. S., et al. Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone. Neural. Dev. 7, 25 (2012).
  26. Powrozek, T. A., Olson, E. C. Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons. Alcohol. , (2012).
  27. Matsuda, T., Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101, 16-22 (2004).
  28. Suter, B., Nowakowski, R. S., Bhide, P. G., Caviness, V. S. Navigating neocortical neurogenesis and neuronal specification: a positional information system encoded by neurogenetic gradients. The Journal of Neuroscience : The Official Journal of The Society for Neuroscience. 27, 10777-10784 (2007).
  29. Englund, C., et al. and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. J. Neurosci. 25, 247-251 (2005).
  30. Kwon, G. S., Hadjantonakis, A. K. Eomes::GFP-a tool for live imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryo. Genesis. 45, 208-217 (2007).
  31. Pearlman, A. L., Sheppard, A. M. Extracellular matrix in early cortical development. Prog. Brain Res. 108, 117-134 (1996).
  32. Milev, P., et al. Differential regulation of expression of hyaluronan-binding proteoglycans in developing brain: aggrecan, versican, neurocan, and brevican. Biochemical and Biophysical Research Communications. 247, 207-212 (1998).
  33. Kwan, K. Y., et al. SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons. Proceedings of the National Academy of Sciences of the United States of America. 105, 16021-16026 (2008).
  34. McKenna, W. L., et al. Tbr1 and Fezf2 regulate alternate corticofugal neuronal identities during neocortical development. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience. 31, 549-564 (2011).
  35. Tabata, H., Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience. 103, 865-872 (2001).
  36. Stancik, E. K., Navarro-Quiroga, I., Sellke, R., Haydar, T. F. Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex. J. Neurosci. 30, 7028-7036 (2010).
  37. Powrozek, T. A., Zhou, F. C. Effects of prenatal alcohol exposure on the development of the vibrissal somatosensory cortical barrel network. Brain Res. Dev. Brain Res. 155, 135-146 (2005).
  38. O'Dell, R., et al. . Society for Neuroscience. , (2011).
  39. Tombol, T., Hajdu, F., Somogyi, G. Identification of the Golgi picture of the layer VI cortic-geniculate projection neurons. Experimental Brain Research. Experimentelle Hirnforschung. Experimentation Cerebrale. 24, 107-110 (1975).
  40. Brumberg, J. C., Hamzei-Sichani, F., Yuste, R. Morphological and physiological characterization of layer VI corticofugal neurons of mouse primary visual cortex. Journal of Neurophysiology. 89, 2854-2867 (2003).
  41. Jones, E. G. Synchrony in the interconnected circuitry of the thalamus and cerebral cortex. Annals of the New York Academy of Sciences. 1157, 10-23 (2009).
  42. Kowalczyk, T., et al. Intermediate Neuronal Progenitors (Basal Progenitors) Produce Pyramidal-Projection Neurons for All Layers of Cerebral Cortex. Cereb. Cortex. , (2009).
  43. Konig, N., Roch, G., Marty, R. The onset of synaptogenesis in rat temporal cortex. Anatomy and Embryology. 148, 73-87 (1975).

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