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Neuroscience

Neonatal Subventricular Zone Electroporation

Published: February 11th, 2013

DOI:

10.3791/50197

1Department of Neurosurgery and Cellular & Molecular Physiology, Yale University School of Medicine

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.

Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

This procedure is in accordance with Yale IACUC requirements. Scientists should make sure that IACUC guidelines are approved and followed according to their institutional requirements.

1. Section 1. Preparation of DNA, Solutions, and Glass Pipettes

  1. Generate high purity (OD 260/280 > 1.80) high concentration (μg/μl > 2.5) endotoxin-free DNA.
  2. Prepare 0.9% saline solution and sterile filter through a 22 μm filter.
  3. Place 10 cm fire polished borosilica.......

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Neonatal subventricular zone electroporation results in the labeling of nearly all radial glia contiguous with the dorsal, dorsal lateral, and lateral subventricular zone following the "sweeping" movement of the tweezer electrode (Figure 1E). However, electroporation can be tailored to the requirement of the respective experiment but not sweeping the electrode and using specific placement and orientation as detailed in the video. For example, since dorsally localized radial glia differ from those .......

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Here we detail the technique of neonatal SVZ electroporation, a technique to rapidly and robustly label and manipulate SVZ stem cells and their progeny. There are several advantages that electroporation has in comparison to other techniques. First, given the focal labeling of cells, one is able to discern cell autonomous and non-cell autonomous effects. Second, genetic manipulation using inducible systems allows one to compare effects prior to or after synaptic integration. Furthermore, one can bypass the use of m.......

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This work was supported by grants from the Department of Defense (Idea development award, W81XWH-10-1-0041, A.B), CT Stem cell grant (A.B.), and a National Institute of Health NRSA 10668225 (D.M.F). The present material is based on work partly supported by the State of Connecticut under the Connecticut Stem Cell Research Grants Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Connecticut, the Department of Public Health of the State of Connecticut or CT Innovations, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparatio....

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
Heavy Polished Borosilicate Tubing Sutter Instrument BF150-110-10
Dual-Stage Glass Micropipette Puller Narishige PC-10H
Fast Green Fischer Scientific 0521192205
ECM 830 Square Wave Pulse generator Harvard Apparatus 45-0052
Tweezertrodes Harvard Apparatus 45-0488
Fiber-Optic Light Source Fisher Scientific 12-562-36
Tungsten Halogen lamp USHIO America, Inc 1002247
Picospritzer II Parker Instruments 052-0312-900

  1. Kriegstein, A., Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annual Review of Neuroscience. 32, 149-184 (2009).
  2. Imayoshi, I., Sakamoto, M., Kageyama, R. Genetic methods to identify and manipulate newly born neurons in the adult brain. Frontiers in Neuroscience. 5, 64 (2011).
  3. LoTurco, J., Manent, J. B., Sidiqi, F. New and improved tools for in utero electroporation studies of developing cerebral cortex. Cereb Cortex. 19, i120-i125 (2009).
  4. 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).
  5. Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. Efficient in vivo electroporation of the postnatal rodent forebrain. PloS ONE. 3, e1883 (2008).
  6. Chesler, A. T., et al. Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis. PloS ONE. 3, e1517 (2008).
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  8. Alvarez-Buylla, A., Kohwi, M., Nguyen, T. M., Merkle, F. T. The heterogeneity of adult neural stem cells and the emerging complexity of their niche. Cold Spring Harbor Symposia on Quantitative Biology. 73, 357-365 (2008).
  9. Fernandez, M. E., Croce, S., Boutin, C., Cremer, H., Raineteau, O. Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis. Neural Development. 6, 13 (2011).
  10. de Chevigny, A., et al. miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons. Nature Neuroscience. 15, 1120-1126 (2012).
  11. Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice. Frontiers in Neuroscience. 4, (2010).
  12. Feliciano, D. M., Quon, J. L., Su, T., Taylor, M. M., Bordey, A. Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion. Human Molecular Genetics. 21, 799-810 (2012).
  13. Iguchi, T., Yagi, H., Wang, C. C., Sato, M. A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique. PloS ONE. 7, e33380 (2012).

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