Name: neonatal pial surface electroporation
Uploaded: 2014-05-07T14:00:00
Description: Watch this Scientific Journal Video about Neonatal Pial Surface Electroporation at JoVE.com

The authors would like to acknowledge support from the Samuel Oschin Comprehensive Cancer Institute Cancer Research Forum Award as well as funds from the Regenerative Medicine Institute of Cedars-Sinai, and the Guerin Family. The project described was supported in the form of a CTSI Core Voucher funded by the National Center for Research Resources, Grant UL1RR033176, and is now at the National Center for Advancing Translational Sciences, Grant UL1TR000124. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

This procedure is in accordance with Cedars-Sinai IACUC requirements. Investigators should ensure institutional IACUC compliance prior to proceeding. All tools and reagents should be sterilized prior to use.
1. Preparation of Tools, Solutions, and DNA Mixture
<ol>
	<li>Insert 100-mm fire polished borosilicate glass capillary tubes into a micropipette puller. Set heating to allow for standard weighted pull to form pipette tip of approximately 17.5 mm. Cut tips with sharp surgical scissors at ...

<ol>
	<li>Breunig, J. J., Haydar, T. F., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells:+historical+perspective+and+future+prospects.">Neural stem cells: historical perspective and future prospects.</a> Neuron. 70 (4), 614-625 (2011).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287 (5457), (2000).</li><li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annu Rev Neurosci. 32, 149-184 (2009).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Costa, M. R., Kessaris, N., Richardson, W. D., Gotz, M., Hedin-Pereira, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginal+zone/layer+I+as+a+novel+niche+for+neurogenesis+and+gliogenesis+in+developing+cerebral+cortex.">The marginal zone/layer I as a novel niche for neurogenesis and gliogenesis in developing cerebral cortex.</a> J Neurosci. 27 (42), 11376-11388 (2007).</li><li>Breunig, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+genetic+targeting+of+pial+surface+neural+progenitors+and+immature+neurons+by+neonatal+electroporation.">Rapid genetic targeting of pial surface neural progenitors and immature neurons by neonatal electroporation.</a> Neural Dev. 7, (2012).</li><li>Ohira, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-induced+neurogenesis+of+neocortical+layer+1+progenitor+cells.">Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells.</a> Nat Neurosci. 13 (2), 173-179 (2010).</li><li>Bystron, I., Blakemore, C., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+human+cerebral+cortex:+Boulder+Committee+revisited.">Development of the human cerebral cortex: Boulder Committee revisited.</a> Nat Rev Neurosci. 9 (2), 110-122 (2008).</li><li>Schmechel, D. E., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Golgi+study+of+radial+glial+cells+in+developing+monkey+telencephalon:+morphogenesis+and+transformation+into+astrocytes.">A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes.</a> Anat Embryol (Berl. 156 (2), 115-152 (1979).</li><li>Halfter, W., Dong, S., Yip, Y. P., Willem, M., Mayer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+function+of+the+pial+basement+membrane+in+cortical+histogenesis.">A critical function of the pial basement membrane in cortical histogenesis.</a> J Neurosci. 22 (14), 6029-6040 (2002).</li><li>Bystron, I., Rakic, P., Molnar, Z., Blakemore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+neurons+of+the+human+cerebral+cortex.">The first neurons of the human cerebral cortex.</a> Nat Neurosci. 9 (7), 880-886 (2006).</li><li>Tanaka, D. H., Maekawa, K., Yanagawa, Y., Obata, K., Murakami, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidirectional+and+multizonal+tangential+migration+of+GABAergic+interneurons+in+the+developing+cerebral+cortex.">Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex.</a> Development. 133 (11), 2167-2176 (2006).</li><li>Ang Jr, E. S., Haydar, T. F., Gluncic, V., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+migratory+coordinates+of+GABAergic+interneurons+in+the+developing+mouse+cortex.">Four-dimensional migratory coordinates of GABAergic interneurons in the developing mouse cortex.</a> J Neurosci. 23 (13), 5805-5815 (2003).</li><li>Tamamaki, N., Fujimori, K. E., Takauji, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+route+of+tangentially+migrating+neurons+in+the+developing+neocortical+intermediate+zone.">Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone.</a> J Neurosci. 17 (21), 8313-8323 (1997).</li><li>Larkum, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin+and+yang+of+cortical+layer+1.">The yin and yang of cortical layer 1.</a> Nat Neurosci. 16 (2), 114-115 (2013).</li><li>Jiang, X., Wang, G., Lee, A. J., Stornetta, R. L., Zhu, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+two+new+cortical+interneuronal+circuits.">The organization of two new cortical interneuronal circuits.</a> Nat Neurosci. 16 (2), 210-218 (2013).</li><li>Endo, T., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+different+AMPA-type+glutamate+receptors+in+morphologically+identified+neurons+in+rat+superficial+superior+colliculus.">Functionally different AMPA-type glutamate receptors in morphologically identified neurons in rat superficial superior colliculus.</a> Neuroscience. 108 (1), 129-141 (2001).</li><li>Schmidt, M., Ozen Boller, M., G, W. C., Hall, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disinhibition+in+rat+superior+colliculus+mediated+by+GABAc+receptors.">Disinhibition in rat superior colliculus mediated by GABAc receptors.</a> J Neurosci. 21 (2), 691-699 (2001).</li><li>Ackman, J. B., Burbridge, T. J., Crair, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+waves+coordinate+patterned+activity+throughout+the+developing+visual+system.">Retinal waves coordinate patterned activity throughout the developing visual system.</a> Nature. 490 (7419), 219-225 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472 (7343), 351-355 (2011).</li><li>Boller, M., Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+maturation+of+GABA(A)+and+GABA(C)+receptor+function+in+the+mammalian+superior+colliculus.">Postnatal maturation of GABA(A) and GABA(C) receptor function in the mammalian superior colliculus.</a> Eur J Neurosci. 14 (8), 1185-1193 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li><li>De Vry, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+the+central+nervous+system:+a+non-viral+approach+for+targeted+gene+delivery.">In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery.</a> Prog Neurobiol. 92 (3), 227-244 (2010).</li><li>Stancik, E. K., Navarro-Quiroga, I., Sellke, R., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+ventricular+zone+neural+precursors+contributes+to+neuronal+fate+diversity+in+the+postnatal+neocortex.">Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex.</a> J Neurosci. 30 (20), 7028-7036 (2010).</li><li>De la Rossa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+circuit+connectivity+in+postmitotic+neocortical+neurons.">In vivo reprogramming of circuit connectivity in postmitotic neocortical neurons.</a> Nat Neurosci. 16 (2), 193-200 (2013).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3 (4), (2008).</li><li>Chesler, A. T., Le Pichon, C. E., Brann, J. H., Araneda, R. C., Zou, D. J., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+gene+expression+by+postnatal+electroporation+during+olfactory+interneuron+nurogenesis.">Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis.</a> PLoS One. 3 (1), (2008).</li><li>dal Maschio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+and+site-directed+in+utero+electroporation+by+a+triple-electrode+probe.">High-performance and site-directed in utero electroporation by a triple-electrode probe.</a> Nat Commun. 3, (2012).</li><li>Yoshida, A., Yamaguchi, Y., Nonomura, K., Kawakami, K., Takahashi, Y., Miura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes Cells. 15 (5), 501-512 (2010).</li><li>Chen, F., LoTurco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+stable+transgenesis+of+radial+glia+lineage+in+rat+neocortex+by+piggyBac+mediated+transposition.">A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.</a> J Neurosci Methods. 207 (2), 172-180 (2012).</li><li>Feliciano, D. M., Lafourcade, C. A., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+subventricular+zone+electroporation.">Neonatal subventricular zone electroporation.</a> J Vis Exp. , (2013).</li><li>Lam, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nat Methods. 9 (10), 1005-1012 (2012).</li><li>Subach, O. M., Cranfill, P. J., Davidson, M. W., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+monomeric+blue+fluorescent+protein+with+the+high+chemical+stability+of+the+chromophore.">An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore.</a> PLoS One. 6 (12), (2011).</li></ol>

The most critical aspect for successful electroporation of pial surface progenitors are: 1) targeting of plasmid mix to the pial surface; 2) avoiding the generation of hematomas at the injection site; and 3) avoiding mortality associated with midbrain electroporation.
Appropriately targeting the pial surface is accomplished by measured and careful puncturing of the skull to avoid penetration of the pial surface. Improper targeting to the overlying skin or underlying ventricle or brain parenchy...

Neural stem and progenitor cells are present throughout the mammalian CNS1,2. Their nature and properties in embryonic and adult germinal zones surrounding the ventricular regions of the brain and spinal cord have been extensively documented in the past decade1-3. In large part, this has been due to the development of increasingly precise genetic tools, such as nervous system specific Cre recombination of floxed alleles or retroviral lineage tracing4. However, one progenitor region&#8212;the pial surface progenitor zone&#8212;has only recently been described in any detail5-7&#160;and awaits comprehensive examination.
The pial surface of the brain is defined as the interface between the surface of the brain and the surrounding meninges8. During development, neuroepithelial and, later, radial glial end feet attach to this surface9,10. Some of the first neurons in the human brain and many neuronal mitoses are observed in this region11. Later, during embryonic neurogenesis, cortical interneurons are known to traverse the pial region, in addition to their migratory routes in the intermediate zone and subventricular zone12-14. During this period, stem cells can be cultured from this zone and it appears to be an active site of neuro- and gliogenesis5. In the adult brain, it has been reported that interneurons can be born from pial surface progenitors following hypoxic challenge7. However, the contribution of this region to his to genensis during embryonic and postnatal development has remained obscure in part due to the difficulty of specifically investigating this region6. In the superior colliculus and in the cerebral cortex, superficial (or layer I in the cortex) interneurons may modulate the circuit output of underlying excitatory neuron populations and thus contribute significantly to the function of these structures. In particular, layer 1 interneurons are in prime position to regulate the firing of neurons throughout the upper layers of the cerebral cortex given their extensive connectivity to the superficial and deep layers of cortical columns15,16. In a similar manner, horizontal interneurons receive excitatory input from cortical and retinal fibers, project over a relatively wide area and are speculated to mediate inhibition of neuronal populations responding to remote visual stimuli17,18. Also, their morphology is well-suited to play a potential role in the patterned wave activity in the developing visual system19. Interestingly, interneuron development and maturation happens to a large degree postnatally. Further, this maturation process has been found to be regulated by neuronal activity and is therefore a substrate of developmental plasticity with lifelong consequences on circuit function20,21. Notably, no promoters are described which can specifically target these cells transgenically. Dividing progenitors can be targeted with retrovirus7&#160;but virus production is time consuming and requires skill to yield the high titers needed for cell transduction.
Electroporation has led to a renaissance in the study of neurodevelopment as it allows for rapid and efficient genetic interrogation of signaling pathways in neural progenitors4,22,23. Electroporation involves the injection of plasmid DNA, followed by the delivery of electric pulses to the outside of the head, to unidirectionally drive the DNA into the proliferating progenitors surrounding the ventricles4,22,23. Electroporation appears to require transit of cells through M phase of the cell cycle for expression of plasmid transgenes24. Specifically, it has been found that only cells passing through M phase within 8 hr&#160;of electroporation of plasmids will express transgenes despite their effective delivery to all cells within ~160 &#181;m of the ventricular wall24. It is speculated that this is due to the need for nuclear envelope breakdown in allowing for nuclear access of the episomal plasmids, as chemicals causing nuclear permeabilization can induce expression of plasmids in post mitotic cells25. Originally employed in the embryo22, electroporation was adapted for use in the postnatal brain much later26,27. Recently, we have adapted electroporation for use in the genetic manipulation of pial surface progenitors6. Further, using this approach we have shown that there are apparently two distinct lineages of progenitors in this region&#8212;interneuronal and astrocytic6. This protocol details a simple, rapid, and powerful way to target these cells for the interrogation of the mechanisms regulating development of these cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:695px;" width="694">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Item Name</td>
			<td style="width:269px;">Vendor</td>
			<td style="width:177px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fire Polished Borosilicate Tubing</td>
			<td>World Precision Instruments, Inc.</td>
			<td>1B100F-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Puller</td>
			<td>Sutter Instruments Company</td>
			<td>P-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green FCF</td>
			<td>Sigma Aldrich, Inc.</td>
			<td>F7528</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XenoWorks Digital Microinjector</td>
			<td>Sutter Instruments Company</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECM 830 Generator</td>
			<td><a name="RANGE!B6">Harvard Apparatus, BTX Instrument Div</a></td>
			<td>45-0052</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mm Platinum Tweezertrodes</td>
			<td>Harvard Apparatus, BTX Instrument Div</td>
			<td>45-0487</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SignaGel Electrode Gel</td>
			<td>Cardinal Health</td>
			<td>70315-025</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tris-EDTA Buffer, pH 8.0</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>11-01-02-05</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infrared Heat Lamp</td>
			<td>VWR</td>
			<td>36547-009</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Scissors Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
		</tr>
	</tbody>
</table>

neonatal pial surface electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

Pial surface electroporation results in the expression of plasmid DNA in cells&#8212;mostly progenitors&#8212;at or near the pial surface6. More specifically, the orientation of the electrodes is critical in dictating the direction of plasmid movement and subsequent expression. Thus, in dual electrode configurations, the plasmid is directed in roughly a straight vector between the negative and positive electrode. Therefore, if the negative pole is placed over the injection site and the positive pole is ventral...

Watch this Scientific Journal Video about Neonatal Pial Surface Electroporation at JoVE.com

Neonatal Pial Surface Electroporation

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...