Name: ex utero electroporation and organotypic slice culture of mouse hippocampal tissue
Uploaded: 2015-03-04T15:00:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

NOTE: All animal experiments were carried out in accordance with the German law and were approved by the government offices in T&#252;bingen.
1. Preparation of Micropipettes, Solutions and Membranes
<ol>
	<li>Preparation of Micropipettes
	<ol>
		<li>Pull glass micropipettes using a micropipette puller with the following program: Heat: 540, Pull: 125, Velocity: 20 and Delay: 140. The needle length amounts to 5.5 cm.</li>
		<li>Bevel needles using a microgrinder to obtain a suitable tip size of 4 mm. Store the needles in a box or 15 cm Petri dish to prevent damaging of the tips.</li>
	</ol>
	</li>
	<li>Preparation of Solutions
	<ol>
		<li>Plasmid DNA Solution
		<ol>
			<li>Prepare plasmid DNA containing the desired cDNA construct using an Endotoxin free Maxi-prep kit according to manufacturer&#8217;s protocol.</li>
			<li>Adjust plasmid DNA solution to a final concentration of 3 &#181;g/&#181;l (without GFP spike vector) or 4 &#181;g/&#181;l (with 1 &#181;g of GFP spike vector) in endotoxin free Tris-EDTA buffer containing Fast Green (final concentration 0.05%).</li>
		</ol>
		</li>
		<li>Laminin Stock solution
		<ol>
			<li>Dissolve 1 mg of laminin in sterile water to a final volume of 1 ml. Prepare 100 &#181;l aliquots and store at -80 &#176;C.</li>
		</ol>
		</li>
		<li>Poly-L-Lysine Stock Solution
		<ol>
			<li>Dissolve 50 mg of poly-L-lysine in 50 ml of sterile water to a final concentration of 1 mg/ml. Prepare 1 ml aliquots and store in -20 &#176;C.</li>
		</ol>
		</li>
		<li>Complete Hank&#8217;s Balanced Salt Solution (Complete HBSS)
		<ol>
			<li>Prepare Complete HBSS by combining 100 ml of 10x HBSS, 2.5 ml of 1 M HEPES buffer (pH 7.4), 30 ml of 1 M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1 M NaHCO3. Add sterile water up to 1 L and store at 4 &#176;C. 
			NOTE: Autoclave all solutions expect 1 M HEPES buffer and 1 M D-glucose, which are filter sterilized.</li>
		</ol>
		</li>
		<li>Slice Culture Medium
		<ol>
			<li>Prepare slice culture medium by adding 35 ml of Basal Medium Eagle, 12.9 ml of complete HBSS (1.2.4), 1.35 ml of 1 M D-glucose, 250 &#181;l of 200 mM L-glutamine, and 500 &#181;l of penicillin-streptomycin to obtain a final volume of 50 ml. Add horse serum to a final concentration of 5% and store at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Low-Melting Point (LMP) Agarose
		<ol>
			<li>Prepare a 4% LMP agarose solution by adding 2 g of LMP agarose to 50 ml of complete HBSS (1.2.4) followed by heating in a microwave for 1-2 min at high power. Keep this solution in a water bath at 37 &#8211; 39 &#176;C. Store the solution at 4 &#176;C and reuse.</li>
		</ol>
		</li>
		<li>Paraformaldehyde Solution
		<ol>
			<li>In a fume hood prepare a 4% paraformaldehyde (PFA) solution by adding 4 g of paraformaldehyde to 100 ml of 1x PBS. Heat the solution to 60 &#176;C and add a few drops of 1 N NaOH until the solution becomes clear.</li>
		</ol>
		</li>
		<li>Permeabilization Solution
		<ol>
			<li>Dissolve 9 g of BSA in 300 ml of 1x PBS containing 0.3% Trition X-100 and store at 4 &#176;C. Add 10% sodium azide for long term storage.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Coating of Membrane Inserts
	<ol>
		<li>Dilute one aliquot of laminin stock solution (1.2.2) and one aliquot of poly-L-lysine stock solution (1.2.3) in sterile water to a final volume of 12 ml.</li>
		<li>Place membrane inserts into 6 well plates with each well containing 2 ml of sterile water. Add 1 ml of coating solution on top of the membrane and incubate O/N at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>After incubation wash the membrane inserts three times with 1 ml of sterile water and dry. Use coated membrane inserts on the same day or store at 4 &#176;C for up to four weeks in a dry 6 well plate.</li>
	</ol>
	</li>
</ol>
2. DNA Injection and Electroporation of E15.5 and E18.5 Embryos
<ol>
	<li>Anesthetize a time mated female mouse by placing it into an anesthetizing chamber saturated with 5% isoflurane and connected to a vaporizer. Circulate isoflurane and oxygen at a rate of 1 L/min. Keep the animal in the box for 2-4 min or until unconscious which is tested by pinching between the paws of the mouse.</li>
	<li>Euthanize the unconscious mouse by cervical dislocation on embryonic day (E) 15.5 or 18.5. Dissect the uterus containing the embryos13 and place it into a Petri dish containing 15-20 ml of cold complete HBSS. 
	NOTE: From this point onwards, keep the embryos and tissues on ice.</li>
	<li>Use a pair of scissors to separate each embryo from the uterine horn and place into a second Petri dish containing cold complete HBSS.</li>
	<li>Under a dissecting microscope, sever the uterine muscle wall and the placenta using a pair of fine forceps (#55) and scissors. Carefully release the embryo from the yolk sac.</li>
	<li>Use a pair of Bonn scissors to decapitate the embryos just above the forelimbs at a 60&#176; angle. If the experiment requires genotyping of the embryos, collect a tissue sample for genomic DNA isolation (a small piece of tail).</li>
	<li>Transfer the head to a clean and dry Petri dish. Because the head had been decapitated in a 60&#176; angle, the head should tilt to one side when placed dorsal side up.</li>
	<li>Place a needle carefully into the middle of the hemisphere close to the bregma (Figure 1A, B). Inject approximately 2-3 &#181;l (at 3 or 4 &#181;g/&#181;l) of DNA solution by stepping on the pedal of the picospritzer III using 30 pounds of pressure for the duration of 10-15 msec per pulse, applying 5-8 pulses. The duration and number of the pulses depends on the diameter of the needle opening with smaller openings requiring more time. The interval between each pulse amounts to 1 sec.</li>
	<li>Before placing the electrodes, apply a few drops of complete HBSS on the head of the embryo. Place the electrodes in such a way that the &#8216;negative&#8217; terminal is on the same side as the injected ventricle and the &#8216;positive&#8217; electrode on the opposite side of the injected ventricle below the ear of the embryo&#8217;s head (Figure 1C, D). Apply 5 pulses of 50 V.
	<ol>
		<li>Use 3 mm electrodes for E 15.5 and 5 mm electrodes for E 18.5.</li>
	</ol>
	</li>
</ol>
3. Dissection of the Brain
<ol>
	<li>After the electroporation, peel off the skin from the head with the help of a pair of fine forceps. Using a pair of spring scissors make a small incision in the middle of the cerebellum at the midline of the skull.</li>
	<li>Insert the spring scissors into the incision and cut longitudinally along the sagittal suture. Peel off the skull and detach the brain from the skull by using fine forceps. Transfer the whole brain into 15-20 ml cold complete HBSS solution.</li>
	<li>Meanwhile, pour 4% LMP agarose, kept at 37-39 &#176;C in a water bath, into a peel-away mold.</li>
	<li>Take the brain out of the complete HBSS by a small scoop spatula and drain excess of HBSS by using fine tissue paper or Kimwipes.</li>
	<li>Place the whole brain gently into the agarose and adjust its position with a fine needle. Keep the mold on ice until the agarose is solidified and the block is sectioned (for coronal sections the olfactory bulbs point up).</li>
</ol>
4. Vibratome Sectioning and Slice Culture
<ol>
	<li>Trim the LMP agarose blocks and glue them to the specimen stage using &#8216;super glue&#8217;. After the glue is dry transfer the specimen stage to the buffer tray of the vibratome and fill with ice cold complete HBSS until the block is immersed in the solution. 
	NOTE: Sterilize all instrument and equipment surfaces with 70% ethanol before sectioning.</li>
	<li>Prepare 250 &#181;m thick vibratome sections using a new blade as followed.
	<ol>
		<li>Trim the block with the following settings; frequency &#8211; 60 Hz, amplitude &#8211; 0.7 &#181;m, speed 16-18 mm/sec. Cut the sections containing the desired tissue with the above settings at slow speed (9 mm/sec). Starting the sectioning from the hindbrain, collect 5-7 sections from the cerebrum.</li>
		<li>Transfer the sections to a clean 6 well culture dish containing 5 ml of ice cold complete HBSS with the help of a bent spatula and keep on ice till all the sections are collected (Figure 1E).</li>
	</ol>
	</li>
	<li>Moisten the membrane in a cross fashion with 100 &#181;l of complete HBSS before placing the sections on the membrane, to facilitate the orientation of the sections.</li>
	<li>Transfer the sections using a bent spatula onto the membrane (pick up a corner of the section with forceps and pull onto the spatula and then use the forceps to push the section onto the membrane). Place up to five sections on one membrane and arrange by using forceps (Figure 1F). Do not overlap the sections with each other.
	<ol>
		<li>Take the excess of HBSS off the membrane using a pipette. The specific membranes used here are attached to a frame and inserted into the tissue culture plate, which allows the tissue to be in contact but not covered by the medium.</li>
	</ol>
	</li>
	<li>Place the membrane inserts into a 6 well plate containing 1.8 ml of slice culture medium (1.2.5) (Figure 1G, H). Incubate the culture dish at 37 &#176;C with 5% CO2 for 11 DIV or 14 DIV. Change half the medium (0.9 ml) every second day. 
	NOTE: At this stage, add reagents like Bromodeoxyuridine (BrdU; 10 &#181;M final concentration) for labeling proliferating cells to the media for the first 20 hr of the culture time.</li>
</ol>
5. Fixation of the Sections Followed by Immunofluorescence Staining
<ol>
	<li>Use a clean and sharp scalpel blade to cut and trim the membranes depending on the orientation of the sections.</li>
	<li>Transfer the sections along with the membrane to a 24 well plate containing 1 ml of 4% PFA (1.2.7). Incubate the sections for 1 hr at RT followed by 3 washes with 1x PBS for 15 min each. Incubate the sections O/N with permeabilization solution at 4 &#176;C with gentle agitation.</li>
	<li>The following day, incubate the sections with appropriate primary antibodies, diluted in permeabilization solution, O/N or for 48 hr at 4 &#176;C with gentle agitation.</li>
	<li>Wash the sections 3 times for 15 min with 1x PBS and incubate O/N at 4 &#176;C with the appropriate secondary antibodies diluted in permeabilization solution.</li>
	<li>After the incubation with secondary antibodies, wash the sections once with 1x PBS for 15 min followed by DAPI staining for 10 min.</li>
	<li>Wash the sections 3 times with 1x PBS for 15 min each and transfer to microscope slides. Add ImmunoMount and gently place a coverslip on top of the sections. Dry the slides O/N at 4 &#176;C and seal with nail polish. 
	NOTE: Keep the slides always at 4 &#176;C.
	<ol>
		<li>Analyze the slice cultures by confocal microscopy (Figure 1I).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> Trends Neurosci. 27, 447-452 (2004).</li><li>Frotscher, M., Zhao, S., Forster, E. <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+cell+and+fiber+layers+in+the+dentate+gyrus.">Development of cell and fiber layers in the dentate gyrus.</a> Prog Brain Res. 163, 133-142 (2007).</li><li>Muramatsu, R., Ikegaya, Y., Matsuki, N., Koyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatally+born+granule+cells+numerically+dominate+adult+mice+dentate+gyrus.">Neonatally born granule cells numerically dominate adult mice dentate gyrus.</a> Neuroscience. 148, 593-598 (2007).</li><li>Li, G., Pleasure, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+dentate+gyrus+morphogenesis.">Genetic regulation of dentate gyrus morphogenesis.</a> Prog Brain Res. 163, 143-152 (2007).</li><li>Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+chromatin+immunoprecipitation.">The current state of chromatin immunoprecipitation.</a> Mol Biotechnol. 45, 87-100 (2010).</li><li>Simon, R., 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=A+dual+function+of+Bcl11b/Ctip2+in+hippocampal+neurogenesis.">A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis.</a> Embo J. 31, 2922-2936 (2012).</li><li>Pilaz, L. J., Silver, D. 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(2), (2007).</li><li>Sugiyama, T., Osumi, N., Katsuyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+germinal+matrices+in+the+developing+dentate+gyrus+are+composed+of+neuronal+progenitors+at+distinct+differentiation+stages.">The germinal matrices in the developing dentate gyrus are composed of neuronal progenitors at distinct differentiation stages.</a> Dev Dyn. 242, 1442-1453 (2013).</li><li>Lechler, T., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desmoplakin:+an+unexpected+regulator+of+microtubule+organization+in+the+epidermis.">Desmoplakin: an unexpected regulator of microtubule organization in the epidermis.</a> J Cell Biol. 176, 147-154 (2007).</li><li>Nichols, A. J., O'Dell, R. S., Powrozek, T. A., Olson, E. 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The authors declare that they have no competing financial interests.

The hippocampus has an important function in learning and memory. The dentate gyrus is also one of two brain regions where neurogenesis occurs not only during development but also throughout adulthood. Postnatal and adult hippocampal neurogenesis proceeds in a similar way involving many common factors. Defining the regulatory mechanisms of these factors will be very helpful in understanding neurodegenerative diseases which in turn will lead to new therapies and preventive measures. To obtain this information one requires a system to manipulate single cells and observe them in their native environment as demonstrated by ex utero electroporation followed by organotypic slice culture.
Ex utero electroporation was first successfully applied in studying cortex development11,16. To our knowledge examining the role of Bcl11b in hippocampal development is the first describing ex utero electroporation of DNA into dentate gyrus tissue8. We based our protocol on the methods published by the Polleux group studying cortex development11,12. To successfully apply this method to study the dentate gyrus the size and position of the electrodes had to be adjusted. Placing the negative electrode at the cortex near the injection site and the positive electrode at the opposite site below the ear changed the polarity of the electroporation and succeeded in introducing DNA into cells of the dentate gyrus. Placing the electrodes correctly and applying a current of 0.06 to 0.08 mA turned out to be very crucial steps to achieve satisfactory electroporation results. To obtain the most adequate current depends mainly on the correct placing of the electrodes as described above. Additional critical steps of the protocol are injecting the material into the ventricle as well as handling the slices after vibratome sectioning. The brain tissue is not fixed throughout the whole procedure and therefore the tissue is very soft and easily destroyable when transferring from the vibratome onto the membrane for cultivation. Sections also have to be handled with great care when starting the immunostaining. With these modifications and considerations manipulations of single cells of the dentate gyrus were successfully performed answering questions concerning Bcl11b regulation of early hippocampus development (Figure 2 and 3; this figures have been modified from8).
As mentioned above manipulating single cells and observe their fate is the major advantage of ex utero electroporation. A major drawback of the ex utero approach in comparison with in utero electroporation is the limited time to keep slices in culture. It is also possible that ex utero cultivation conditions might cause a delay in development. In our experiments so far we were able to keep the slices in culture up to 18 days which corresponds to the age of P14. Cultivation of organotypic slices beyond this time lead to disintegration of the tissue. An additional disadvantage of the ex utero approach is the limited number of embryos that can be electroporated per mother. In contrast to in utero electroporation where up to 8 embryos can be treated this number is limited to 4 embryos in the ex utero approach. Dissection of the embryos, electroporation, vibratome sectioning and transfer of the slices into culture conditions have to be performed in a short time to ensure successful slice cultures.
Because major events of dentate gyrus development occur between P14 and P30 it would be necessary to keep organotypic slices in culture for prolonged time periods. In a first attempt to extend the culture time electroporations were performed at E 18.5 adjusting the conditions accordingly (Figure 4; see protocol). Electroporating at this time point allows keeping the brain sections in culture up to P17 or 18. In addition the hippocampal formation is further developed at E 18.5 when compared to E 15.5, e.g., hippocampal structures are already formed and more easily recognizable. In the future we would like to perform ex utero electroporation at even later time points, e.g., P0 to P4 to examine changes during dentate gyrus development up to P30.

The hippocampus plays an important role in memory and learning as well as emotional behavior. One main function consists of the consolidation of short-term memory into long-term memory, which requires high plasticity of the nervous system. The dentate gyrus of the hippocampus acts as the primary gateway for input information and is also one of two brain regions with ongoing neurogenesis throughout adulthood1,2. The development of the hippocampal structure occurs during late embryogenesis and particularly during the first 3 to 4 weeks postnatal3. During early development of the dentate gyrus a stem cell pool is established required for postnatal as well as adult neurogenesis4. Developing neurons pass through various stages, from the stem cell through several stages of progenitor cells to the immature and finally the mature neuron during postnatal as well as adult neurogenesis. At different stages of neurogenesis the expression of specific genes is required to allow the maturation and integration of new neurons into the hippocampal circuitry5,6.
Using mouse genetics and phenotype analysis by immunohistochemistry as well as molecular methods allowed defining the expression pattern and function of many of these genes. In addition microarray analysis as well as chromatin immunoprecipitation (ChIP) provided information about potential direct and indirect target genes7,8. However, there are still many open questions concerning the regulatory mechanisms of hippocampal development, in particular the development of the dentate gyrus. To gain further insight how specific genes are regulated a system is required allowing the manipulation of a small number of cells by down- or up-regulation of the gene of interest and/or its target genes and follow their fate during development. In utero electroporation of shRNAs, cDNA of genes of interest or Cre recombinase provides such a tool. To ensure the presence of the desired DNA or small RNAs expression plasmids should be used for electroporation. This approach is very successfully implemented in studying cortical development9,10, but is a more challenging approach examining the development of the dentate gyrus due to the position of the hippocampal structures in deeper brain layers.
Ex utero electroporation followed by organotypic slice culture is one approach to circumvent this problem11,12. In contrast to in utero electroporation not the whole embryo but only the head is used allowing therefore to place the electrodes in a more favorable way to direct the shRNA/DNA towards the hippocampus and dentate gyrus. Our group successfully employed ex utero electroporation to study the role of the transcription factor Bcl11b during dentate gyrus development8. Bcl11b has a dual role in dentate gyrus development by regulating progenitor cell proliferation as well as differentiation as was demonstrated by immunohistochemistry. To further define a mechanism for Bcl11b involvement in these processes, protocols of the Polleux group11,12 were adjusted to study the dentate gyrus as described below in the protocol section. In a first approach the question was addressed whether Bcl11b is regulating neuronal cell differentiation cell autonomously. A second approach examined whether Desmoplakin, a direct target gene of Bcl11b, is sufficient to rescue the Bcl11b phenotype.

<table><tbody><tr><td>Flaming/ Brown Micropipette Puller</td><td>Sutter Instruments Company (USA)</td><td>P-97</td><td></td></tr><tr><td>Fine Glass Pipettes</td><td>Warner Instruments</td><td>G100F-4</td><td></td></tr><tr><td>Microgrinder</td><td>Narishige, Japan</td><td>EG-44</td><td></td></tr><tr><td>Anesthetic Bracket unit</td><td>Harvard Apparatus</td><td>PY2 34-0412</td><td></td></tr><tr><td>Halovet Vaporizer</td><td>Harvard Apparatus</td><td>PY2 34-0398</td><td></td></tr><tr><td>Fluovac System</td><td>Harvard Apparatus</td><td>PY2 34-0387</td><td></td></tr><tr><td>IMS Fluosorber</td><td>Harvard Apparatus</td><td>PY2 34-0415</td><td></td></tr><tr><td>Anesthetizing Chamber</td><td>Harvard Apparatus</td><td>PY2 34-0460</td><td></td></tr><tr><td>Electroporator</td><td>BEX Company</td><td>CUY21 EDIT</td><td></td></tr><tr><td>Tweezers with disk electrodes</td><td>BEX Company</td><td>LF650P3</td><td>3 mm electrodes for E15.5</td></tr><tr><td>Tweezers with disk electrodes</td><td>BEX Company</td><td>LF650P5</td><td>5 mm electrodes for E18.5</td></tr><tr><td>Picospritzer III</td><td>Parker Hannifin Corporation</td><td>P/N 052-0500-900</td><td></td></tr><tr><td>HM 650 V Vibrating Blade Microtome, 230 V</td><td>Thermo Scientific</td><td>920120</td><td></td></tr><tr><td>Dissection Microscope</td><td>Carl Zeiss Microscopy Gmbh</td><td>Stemi SV8</td><td></td></tr><tr><td>Inverted Microscope</td><td>Leica</td><td>Leica DM IL LED</td><td></td></tr><tr><td>Confocal Microscope</td><td>Leica</td><td>Sp5II</td><td></td></tr><tr><td>6 well dish</td><td>BD Falcon</td><td>#353502</td><td></td></tr><tr><td>6 well dish</td><td>CELLSTAR</td><td>#657160</td><td></td></tr><tr><td>Tissue culture inserts</td><td>BD Falcon</td><td>#353090</td><td></td></tr><tr><td>Fast Green</td><td>Sigma</td><td>F7252</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>#L2020</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>#P5899</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Forceps</td><td>Dumont #55</td><td>11255-20 Inox</td><td></td></tr><tr><td>HBSS 10x</td><td>Life Technology</td><td>14180-046</td><td></td></tr><tr><td>BME</td><td>Life Technology</td><td>41010-26</td><td></td></tr></tbody></table>

ex utero electroporation and organotypic slice culture of mouse hippocampal tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ablation of the transcription factor Bcl11b causes the impairment of progenitor cell proliferation and neuronal differentiation resulting in a reduced dentate gyrus size and cell number. Furthermore mutant neurons fail to integrate into the hippocampal circuitry causing learning and memory impairment8. To answer questions concerning the regulatory mechanism(s) of Bcl11b in these processes ex utero electroporation was employed.
Addressing the question whether Bcl11b cell-autonomously regulates neuronal cell differentiation, mosaic deletions of Bcl11b were generated by ex utero electroporation of a GFP-Cre recombinase construct or GFP alone11 into Bcl11bflox/flox hippocampi at E15.5 followed by organotypic slice culture up to 18 days after electroporation (Figure 2A, B; this Figure has been modified from8). To determine whether Bcl11b regulates differentiation of granule cells cell-autonomously immunofluorescence staining was performed using specific antibodies recognizing NeuroD as well as GFP. NeuroD is expressed in mitotic stages 2b/3 and early postmitotic cells14. We have shown previously that the number of NeuroD positive cells is significantly increased in Bcl11b conditional mutants indicating an arrest of neuronal differentiation8. While the number of GFP positive cells alone and GFP/NeuroD positive cells did not differ in control and mutant cells a significant increase in NeuroD positive cells was observed in the dentate gyrus where cells had received Cre recombinase (Figure 2C; this figure has been modified from8). Finding NeuroD positive cells not only in cells which had received Cre recombinase but also in wild-type cells suggested that indirect mechanisms are involved in Bcl11b regulation of neuronal cell differentiation. From these data, however, additional cell-autonomous functions of Bcl11b cannot be excluded.
Previously, Desmoplakin was determined as a direct target gene of Bcl11b8. It was also shown that Desmoplakin is involved in the regulation of progenitor cell proliferation and differentiation of keratinocytes15. To demonstrate whether Desmoplakin is involved in the regulation of progenitor cell proliferation and/or neuronal differentiation of the dentate gyrus plasmid DNA expressing GFP alone or GFP and Desmoplakin under the control of the CMV promoter was electroporated into control and Bcl11b mutant brains (Figure 3A-C; this figure has been modified from8). The brain slices were cultured for the first 20 hr in the presence of BrdU (10 &#181;M final concentration). At day 11 after electroporation the slice cultures were fixed followed by BrdU immunostaining and the number of BrdU positive cells was determined. Bcl11b mutant tissue electroporated with GFP only contained significantly fewer BrdU positive cells when compared to the control tissue. However, co-electroporation of GFP and Desmoplakin rescued the number of BrdU positive cells to control levels (Figure 3D; this figure has been modified from8). Taken together these data further confirm Desmoplakin as a direct target gene of Bcl11b and its role in the regulation of progenitor cell proliferation.
<img alt="Figure 1" src="/files/ftp_upload/52550/52550fig1highres.jpg" /> 
Figure 1.&#160;Set-up of ex utero electroporation and organotypic slice culture at E15.5. (A) Injection of DNA into one hemisphere. (B) Schematic drawing of DNA injection. (C) Positioning of the electrodes. (D) Schematic drawing of electrode placement. (E) Vibratome sectioning and handling of brain sections. (F) Placing brain sections on specific membranes. (G-I) Bright-field (G) and fluorescence (H, GFP) analysis of slice cultures on day 1 after electroporation. (I) Confocal image of the dentate gyrus at day 11 after electroporation using DAPI and GFP staining. Dashed line indicates the dentate gyrus. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52550/52550fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52550/52550fig2highres.jpg" /> 
Figure 2.&#160;Mosaic deletion of Bcl11b by ex utero electroporation and organotypic slice culture (modified from 8). Electroporation of control vector pCIG2 alone (A) and pCIG2-Cre construct (B) into the dentate gyrus at E15.5 followed by organotypic slice culture for 18 days. Sections were immunostained by using antibodies recognizing GFP (green) and NeuroD (red). Insets display GFP (green) and Bcl11b (red) staining at higher magnification to demonstrate loss of Bcl11b expression in cells expressing Cre-recombinase. (C) Statistical analysis of GFP, NeuroD and GFP/NeuroD positive cells. Dashed lines indicate the dentate gyrus. t-test, *p &#60;0.005; error bars, s.d.; n = 5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52550/52550fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52550/52550fig3highres.jpg" /> 
Figure 3.&#160;Bcl11b phenotype is rescued by re-introduction of Desmoplakin (modified from 8). GFP only (A, B) as well as GFP and Desmoplakin (C) were electroporated into the dentate gyrus of control (A) and mutant (B, C) brains at E15.5 followed by organotypic slice culture for 11 days. Sections were immunostained by using antibodies recognizing GFP (green) and BrdU (red). (D) Statistical analysis of BrdU positive cells. Dashed lines indicate the dentate gyrus. Insets display Desmoplakin staining (green) at higher magnification. t-test, *p &#60;0.01; error bar, s.e.m; n = 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52550/52550fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52550/52550fig4highres.jpg" /> 
Figure 4.&#160;Ex utero electroporation at E 18.5 followed by organotypic slice culture. Injection of DNA expressing GFP into one hemisphere followed by immunostaining at day 16 after electroporation. (A) DAPI staining; (B) GFP staining; (C) merged image. Dashed line indicates dentate gyrus. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52550/52550fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

ex-utero-electroporation-organotypic-slice-culture-mouse-hippocampal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.5K Views.  University of Ulm. Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Video: Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Neuroscience

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of 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.

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

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 ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Preparation of Organotypic Slice Cultures from a Rat Pup&#39;s Hippocampus

Source: Mosa, A. J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52353">Organotypic Slice Cultures for Studies of Postnatal Neurogenesis.</a> J. Vis. Exp. (2015).
This video demonstrates a method for preparing organotypic hippocampal slice cultures from the brain of a rat pup. It outlines the steps involved in the dissection of the hippocampus, slicing, and culture conditions required to promote cell viability and growth in the hippocampal slices, ultimately generating organotypic slice cultures.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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