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 o...

<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+dentate+gyrus:+what+we+are+learning+from+mouse+mutants.">Morphogenesis of the dentate gyrus: what we are learning from mouse mutants.</a> Dev Neurosci. 27, 93-99 (2005).</li><li>Hsieh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+transcriptional+control+of+adult+neurogenesis.">Orchestrating transcriptional control of adult neurogenesis.</a> Genes Dev. 26, 1010-1021 (2012).</li><li>Li, G., Pleasure, S. 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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...

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.

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			<td height="106" style="height:106px;width:71px;">Name of Reagent/ Equipment</td>
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			<td style="width:64px;">3 mm electrodes for E15.5</td>
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			<td height="106" style="height:106px;width:71px;">Tweezers with disk electrodes</td>
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			<td style="width:63px;">LF650P5</td>
			<td style="width:64px;">5 mm electrodes for E18.5</td>
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			<td style="width:63px;">P/N 052-0500-900</td>
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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-aut...

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

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9.4K visualizações.  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.