We thank Dr. Takahiko Matsuda for providing the pCAG-mCherry construct. YCM is supported by a Career Development Award from the Schweppe Foundation and a grant from the Whitehall Foundation.

1. Supply Preparation (not in video)
<ol>
 <li>Prepare a fresh 1X dilution of Penicillin/Streptomycin (Pen/Strep) in 1X PBS. No more than 50 mL is necessary. Filter with a 0.22 &mu;m syringe filter.</li>
 <li>Prepare injection constructs by combining desired plasmid constructs. We put all constructs in the pCAG-Flag vector with appropriate stoichiometry to a final volume of ~10 &mu;L. Also, pCAG-GFP14 or pCAG-mCherry should be added for tracking electroporation efficiency and electroporated cells. We use pCAG-mCherry for this experiment. Lastly, add 0.22 &mu;m-filtered Fast Green dye into the plasmid DNA mixture at a final concentration of 0.05% to visualize the injection efficiency into embryonic chick midbrains.</li>
</ol>
2. Egg Preparation (in video)
<ol>
 <li>In order to acquire HH stage 11 embryos, eggs need to be incubated at 100 &deg;F (37.8 &deg;C) for about 41 hours after fertilization with 25-40% humidity. Place eggs on their side and mark the top of each with a pencil dash. Place trays in a pre-warmed, humidified incubator. Slowly rock eggs by setting the turning function of incubator platforms to "Automatic." Check the humidity and water level every 24 hours.</li>
 <li>Right before starting the experiment, remove eggs from the incubator and leave at room temperature. This will help to slow down the development and prevent over development.</li>
 <li>Clean the surface of each egg with 70% ethanol and wipe down with a Kimwipe.</li>
 <li>Take two 4 cm pieces of scotch tape and stick them side-by-side on the egg to make a large rectangle over the pencil dash. Smooth the tape against the egg. This is where the window will be made. The tape will prevent small pieces of eggshell from falling off when the window is cut open.</li>
 <li>Use a 10 mL syringe and 18&frac12; gauge needle to draw 5 mL of albumin out of the egg. After piercing the shell, angle the needle away from the yolk as to not disturb or damage the embryo while removing albumin. Clean the needle with 70% ethanol between eggs, but be sure the needle is dry before piercing the next egg.</li>
</ol>
3. Glass Injection Needle Preparation (in video)
<ol>
 <li>Injection needles are made using the following settings on a Flaming/Brown Micropipette Puller (Sutter Instrument Model P-97): Heat 670, Pull 120, Vel 40, Time 165. World Precision Instruments (API) 4 inch 1.0 mm thin wall capillary glass (API TW100F-4) is used to make needles.</li>
 <li>Load a pulled glass needle using a P10 pipette and the long-tip capillary loading pipette tip (Eppendorf 5242956.003). Load 5-10 &mu;L, depending on how many eggs are to be injected. Fill the glass needle slowly, creating a continuous column of plasmid solution absent of air bubbles.</li>
 <li>Place the loaded needle in the needle holder attached to the micromanipulator (Narishige MM3) and the Picospritzer III microinjector. View the needle under a dissection microscope and trim the needle at the point where the green fluid stops. It helps to use a dark background to better visualize the needle. Proper needle trimming comes with practice.</li>
</ol>
4. Midbrain/Neural Tube Injection (in video)
<ol>
 <li>Use spring scissors to cut a 2-2.5 cm diameter window in the egg. Start the window from the previous needle hole made when drawing albumin, and rotate the egg in your hand as you cut. Be sure to clean off the scissors with a Kimwipe saturated in 70% ethanol between eggs.</li>
 <li>View chick embryos under a dissection microscope. It may be necessary to train the eyes. It may help to visualize the embryo by injecting 0.22 &mu;m-filtered 20% India ink in PBS under the chick embryo. However, we have found that this step should be avoided if possible, as it tends to decrease survival.</li>
 <li>Injection works best if the embryo is positioned in parallel to the path of the needle, with the head away from the needle. Slowly pierce the neural tube at a 45 degree angle to the midbrain-hindbrain junction. It is very critical to only fill the neural vesicle representing the midbrain with plasmid DNA and Fast Green dye, without significant diffusion into the forebrain and hindbrain regions. Start injecting with the Picospritzer microinjector's duration set at 25 ms; this may need to be changed depending on needle trimming. In general, use between 10-50 ms.</li>
</ol>
5. Electroporation (in video)
<ol>
 <li>Retrieve the injection needle. Place 3 drops of 1X Pen/Strep in PBS solution in the egg on the embryo.</li>
 <li>Check the settings of the BTX EMC830 electroporator:
<table border="1">
 <tbody>
 <tr>
 <td>VOLTAGE:</td>
 <td>20 V</td>
 </tr>
 <tr>
 <td>PULSE LENGTH:</td>
 <td>25 ms</td>
 </tr>
 <tr>
 <td>#PULSES:</td>
 <td>3</td>
 </tr>
 <tr>
 <td>INTERVAL:</td>
 <td>500 ms</td>
 </tr>
 <tr>
 <td>POLARITY:</td>
 <td>UNIPOLAR</td>
 </tr>
 </tbody>
</table>
</li>
 <li>Place the 2 mm long L-shaped platinum electrodes at the same horizontal level as the bottom of the neural tube, with the embryonic midbrain sitting in the middle between two 3 mm apart electrodes. Aligning the electrodes with the bottom of the neural tube is critical for efficient electroporation of the ventral midbrain. To achieve midbrain-specific electroporation, we replaced the original BTX electrodes with two custom-made platinum L-shaped electrodes with short 2 mm long tips. These platinum electrodes are made of 0.5mm diameter platinum wires (Alfa Aesar). These platinum electrodes are just long enough to cover the midbrain of a stage 11 chick embryo, which is about 0.9 mm long, without targeting much of the forebrain or the hindbrain regions. Press the BTX footswitch once. Air bubbles should be generated around the electrodes with each successful electroporation. Carefully remove the electrodes from the egg and wipe the electrodes off with a 70% ethanol-covered Kimwipe. Place another 3 drops of 1X Pen/Strep solution in the egg, on the embryo.</li>
 <li>Cover the window with a 5 cm x 5 cm piece of clear packaging tape. Be sure to seal the egg well and that the egg contents do not come in contact with the tape.</li>
</ol>
6. Incubate and Harvest
<ol>
 <li>Place the eggs back into the incubator. Make sure the turning of incubator platforms is OFF. Incubate eggs until the desired HH stage 23 is achieved.</li>
 <li>Harvest living embryos and place them into common PBS-filled Petri dishes for each condition. Screen harvested embryos under a fluorescent dissection microscope, keeping only embryos with mCherry expression.</li>
 <li>Transfer embryos into individual wells of a 24-well dish. Fill each well with 1 mL of freshly made 2% paraformaldehyde (PFA) and allow to fix 2-3 hours at 4 &deg;C.</li>
 <li>Wash fixed embryos three times for 10 minutes each in PBS. Place embryos sequentially in 15% and 30% sucrose until equilibrated. Embryos will initially float in sucrose, but will sink upon equilibration.</li>
 <li>Check embryos under a fluorescent dissection microscope, taking notes on the mCherry expression pattern and level for each embryo. Embed each embryo in OCT. It is very critical to orient embryos properly to generate optimal midbrain cross sections for subsequent analyses (Figure 1).</li>
 <li>Prepare 18 &mu;m thick midbrain sections using a Leica CM3050S cryostat. Analyze midbrain development and dopaminergic neuron differentiation by immunostaining with appropriate cell type markers.</li>
</ol>
7. Representative Results
A diagrammatic representation of electroporating and analyzing embryonic chick midbrain is shown in Figure 1. In successfully injected and electroporated chick embryos, mCherry expression should be visible in the midbrain region after further development (Figure 2A, B, C). Assuming that mCherry correlates with the expression of other co-electroporated genes, embryos exhibiting proper mCherry expression can then be fixed and embedded to prepare midbrain cryosections for further study (Figure 2D). Generally, about 50% of ventral midbrain neural progenitors can be efficiently transfected with the above protocol. The specification of dopaminergic fate in electroporated neural progenitors can be examined by the co-localization of mCherry, Flag-tagged gene, with dopaminergic neuron markers such as Tyrosine Hydroxylase (TH) (Figure 3). The patterning of progenitor domains in ventral midbrain can be investigated by co-immnostaining cryosections with different progenitor domain markers.
Insufficient mCherry expression suggests that ectopic genes are likely not efficiently expressed. Embryos under this condition should not be used for further studies. Similarly, embryos that did not survive to the end of the experimental procedure can't be used for data collection and analysis.
<img alt="Figure 1" src="/files/ftp_upload/4017/4017fig1.jpg" /> 
Figure 1. Illustration of the embryonic chick midbrain in ovo electroporation assay. HH stage 11 embryonic chick embryos are injected with pCAG-mCherry together with genes of interest mixed with fast green to visualize the injection process (A). After filled with DNA constructs, midbrains are electroporated with a square wave electroporator. Embryos are further incubated until desired HH stage 23 is reached. Then mCherry-positive embryos are harvested, fixed, and embedded (B). Midbrain cryosections are prepared with cutting planes indicated by the white line in Figure 1B, and analyzed by immunostaining with midbrain markers such as Tyrosine Hydroxylase (TH) (C). <a href="https://www.jove.com/files/ftp_upload/4017/4017fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4017/4017fig2.jpg" /> 
Figure 2. Preparation of cryosections from embryonic chick midbrain. After 41 hours incubation, HH stage 23 chick embryos expressing mCherry and genes of interest are harvested (A, B and C), fixed with PFA, treated with sucrose, and embedded in OCT for cryosectioning. Chick embryos are carefully oriented to ensure the preparation of midbrain sections. A typical embryo morphology and the cutting plane for obtaining midbrain sections are shown (D). <a href="https://www.jove.com/files/ftp_upload/4017/4017fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4017/4017fig3.jpg" /> 
Figure 3. Analysis of electroporated midbrain sections. Cryosections from embryos with high levels of mCherry expression (A) are prepared and stained with antibodies that recognize the Flag-tagged gene of interest (B) and midbrain dopaminergic neuron marker TH (C). Embryonic midbrains electroporated with pCAG-mCherry and pCAG-Flag empty vector serve as control. <a href="https://www.jove.com/files/ftp_upload/4017/4017fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Wightman, R. M., Robinson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+changes+in+mesolimbic+dopamine+and+their+association+with+'reward'.">Transient changes in mesolimbic dopamine and their association with 'reward'.</a> J. Neurochem. 82, 721-735 (2002).</li><li>Kelley, A. E., Berridge, K. 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+neuroscience+of+natural+rewards:+relevance+to+addictive+drugs.">The neuroscience of natural rewards: relevance to addictive drugs.</a> J. Neurosci. 22, 3306-3311 (2002).</li><li>Moore, D. J., West, A. B., Dawson, V. L., Dawson, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathophysiology+of+Parkinson's+disease.">Molecular pathophysiology of Parkinson's disease.</a> Annu. Rev. Neurosci. 28, 57-87 (2005).</li><li>Dailly, E., Chenu, F., Renard, C. E., Bourin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine,+depression+and+antidepressants.">Dopamine, depression and antidepressants.</a> Fundam. Clin. Pharmacol. 18, 601-607 (2004).</li><li>Sesack, S. R., Carr, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+prefrontal+cortex+inputs+to+dopamine+cells:+implications+for+schizophrenia.">Selective prefrontal cortex inputs to dopamine cells: implications for schizophrenia.</a> Physiol. Behav. 77, 513-517 (2002).</li><li>Ang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+midbrain+dopaminergic+neuron+development.">Transcriptional control of midbrain dopaminergic neuron development.</a> Development. 133, 3499-3506 (2006).</li><li>Andersson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+intrinsic+determinants+of+midbrain+dopamine+neurons.">Identification of intrinsic determinants of midbrain dopamine neurons.</a> Cell. 124, 393-405 (2006).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Series+of+Normal+Stages+in+the+Development+of+the+Chick+Embryo.">A Series of Normal Stages in the Development of the Chick Embryo.</a> J. Morphol. 88, 49 (1951).</li><li>Bellairs, R., Osmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of chick development. , (2005).</li><li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">'Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1, 203-207 (1999).</li><li>Momose, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Muramatsu, T., Mizutani, Y., Ohmori, Y., Okumura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+nonviral+transfection+methods+for+foreign+gene+expression+in+early+chicken+embryos+in+ovo.">Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.</a> Biochem. Biophys. Res. Commun. 230, 376-380 (1997).</li><li>Nishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+in+vivo+gene+transfer+using+intraarterial+plasmid+DNA+injection+following+in+vivo+electroporation.">High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.</a> Cancer Res. 56, 1050-1055 (1996).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 16-22 (2004).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408 (2007).</li><li>Chesnutt, C., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid-based+short-hairpin+RNA+interference+in+the+chicken+embryo.">Plasmid-based short-hairpin RNA interference in the chicken embryo.</a> Genesis. 39, 73-78 (2004).</li></ol>

The authors have no potential conflicting interests to disclose.

The in ovo electroporation of embryonic chick midbrain offers a low cost and rapid alternative to the generation of transgenic or knockout animals to perform in vivo study of gene functions in midbrain dopaminergic neuron development. Using short 2 mm long L-shaped platinum electrodes together with embryonic midbrain-specific DNA injection is the key for achieving efficient expression of the gene of interest in midbrain dopaminergic neuron progenitors. In addition, using correct HH stage 11 chick embryo...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Fertilized chicken eggs</td>
			<td>Charles River Laboratories</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>GQF Manufacturing</td>
			<td>1502 Sportsman</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BTX Electroporator</td>
			<td>BTX Harvard Apparatus</td>
			<td>ECM830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0162 L-shaped genetrodes</td>
			<td>For use with ECM830 electroporator</td>
		</tr>
		<tr>
			<td>Platinum iridium wire</td>
			<td>Alfa Aesar</td>
			<td>#10056</td>
			<td>0.5 mm diameter 
			For making the 2 mm long electrodes</td>
		</tr>
		<tr>
			<td>Glass capillary tube</td>
			<td>World Precision Instrument</td>
			<td>TW100F-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microloader pipette tip</td>
			<td>Eppendorf</td>
			<td>5242 956.003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>India ink</td>
			<td>Staples</td>
			<td>&nbsp;</td>
			<td>Filter 20% before use</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Parker Automation, 
			Parker Hannifin Cooperation</td>
			<td>Picospritzer III</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescent dissection microscope</td>
			<td>Leica</td>
			<td>MZ16F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>D-97</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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Research

JoVE Journal

Neuroscience

11.4K ビュー.  Children's Hospital of Chicago Research Center. 機能や中脳ドーパミン作動性ニューロンの開発中の遺伝子の調節を評価するために、我々は、関与の方法について説明します。エレクトロポレーション。この手法は、脳の発達とドーパミン作動性ニューロンへの分化のさまざまな側面を研究する目?...

ビデオ: In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

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

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...