Name: efficient and cost effective electroporation method to study primary cilium-dependent signaling pathways in the granule cell precursor
Uploaded: 2021-11-30T14:45:00
Description: Watch this Scientific Journal Video about Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor at JoVE.com

This study was supported by HKBU Seed Fund and Tier-2 Start-up Grant (RG-SGT2/18-19/SCI/009), Research Grant Council-Collaborative Research Fund (CRF-C2103-20GF) to C.H.H. Hor.

All animal-related procedures were carried out in compliance with animal handling guidelines and the protocol approved by the Department of Health, Hong Kong. Animal experiment licenses following Animal (Control of Experiments) Ordinance (Cap. 340) were obtained from the Department of Health, Hong Kong Government. The animal work was carried out in compliance with the animal safety ethics approved by HKBU Research Office and Laboratory Safety Committee.&#160;Refer to the Table of Materials for details ab...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+signaling+is+required+for+expansion+of+granule+neuron+precursors+and+patterning+of+the+mouse+cerebellum.">Sonic Hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum.</a> Developmental Biology. 270 (2), 393-410 (2004).</li><li>Wechsler-Reya, R. J., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+neuronal+precursor+proliferation+in+the+cerebellum+by+Sonic+Hedgehog.">Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.</a> Neuron. 22 (1), 103-114 (1999).</li><li>Bangs, F., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+and+mammalian+Hedgehog+signaling.">Primary cilia and mammalian Hedgehog signaling.</a> Cold Spring Harbor Perspectives in Biology. 9 (5), 028175 (2017).</li><li>Hor, C. H. H., Lo, J. C. W., Cham, A. L. S., Leung, W. Y., Goh, E. L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+functions+of+Rab23+on+primary+cilium-+and+Hedgehog+signaling-mediated+cerebellar+granule+cell+proliferation.">Multifaceted functions of Rab23 on primary cilium- and Hedgehog signaling-mediated cerebellar granule cell proliferation.</a> Journal of Neuroscience. 41 (32), 6850-6863 (2021).</li><li>Han, Y. G., 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=Dual+and+opposing+roles+of+primary+cilia+in+medulloblastoma+development.">Dual and opposing roles of primary cilia in medulloblastoma development.</a> Nature Medicine. 15 (9), 1062-1065 (2009).</li><li>Spassky, N., 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=Primary+cilia+are+required+for+cerebellar+development+and+Shh-dependent+expansion+of+progenitor+pool.">Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool.</a> Developmental Biology. 317 (1), 246-259 (2008).</li><li>Wang, T., Larcher, L., Ma, L., Veedu, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+screening+of+commonly+used+commercial+transfection+reagents+towards+efficient+transfection+of+single-stranded+oligonucleotides.">Systematic screening of commonly used commercial transfection reagents towards efficient transfection of single-stranded oligonucleotides.</a> Molecules. 23 (10), 2564 (2018).</li><li>Chicaybam, L., 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=An+efficient+electroporation+protocol+for+the+genetic+modification+of+mammalian+cells.">An efficient electroporation protocol for the genetic modification of mammalian cells.</a> Frontiers in Bioengineering and Biotechnology. 4, 99 (2017).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Chiara Manzini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> Journal of Visualized Experiments: JoVE. (23), e990 (2009).</li><li>Mizoguchi, T., 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=Impaired+cerebellar+development+in+mice+overexpressing+VGF.">Impaired cerebellar development in mice overexpressing VGF.</a> Neurochemical Research. 44 (2), 374-387 (2019).</li><li>Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+of+nerve+cells.">Confocal imaging of nerve cells.</a> Current Laboratory Methods in Neuroscience Research. , 235-247 (2014).</li><li>Corbit, K. C., 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=Vertebrate+Smoothened+functions+at+the+primary+cilium.">Vertebrate Smoothened functions at the primary cilium.</a> Nature. 437 (7061), 1018-1021 (2005).</li></ol>

Transfection of transgenes in primary GCP culture by electroporation method is typically associated with low cell viability and poor transfection efficiency9,10. This paper introduces a cost-effective and reproducible electroporation protocol that has demonstrated high efficiency and viability. In addition, we also demonstrate a straightforward method of studying the primary cilium-dependent Hh signaling pathway in primary GCP cells.
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Cerebellar GCPs are widely used to study the machinery of the Hh signaling pathway in neuronal progenitor cell-types owing to their high abundance and high sensitivity to the Hh signaling pathway in vivo1,2,3,4. In GCPs, the primary cilium acts as a pivotal Hh signal transduction hub5 that orchestrates the proliferation of the precursor cells6,7,8. In vitro visualization of Hh signaling components on the primary cilium is often challenging due to their low endogenous basal levels. Hence, transgene modification of protein expression levels and fluorophore tagging of the gene of interest are useful approaches to study the pathway at molecular resolution. However, genetic manipulation of GCP primary cultures using liposome-based transfection approaches often result in low transfection efficiency, hindering further molecular investigations9. Electroporation increases the efficiency but commonly requires exorbitant vendor-specific and cell type-restricted electroporation reagents10.
This paper introduces a high-efficiency and cost-effective electroporation method to manipulate the Hh signaling pathway components in GCP primary cultures. Using this modified electroporation protocol, a green fluorescent protein (GFP)-tagged Smoothened transgene (pEGFP-Smo) was efficiently delivered to GCPs and achieved high cell survival and transfection rates (80-90%). Furthermore, as evidenced by the immunocytochemical staining, the transfected GCPs showed high sensitivity to Smoothened agonist-induced activation of the Hh signaling pathway by trafficking EGFP-Smo to the primary cilia. This protocol shall be directly applicable and beneficial for experiments that involve in vitro genetic modification of cell types that are difficult to transfect, such as human and rodent primary cell cultures, as well as human induced&#160;pluripotent stem cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GCP Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies LTD</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 70 &#181;m</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I from bovine pancreas</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Earle&#8217;s Balanced Salt Solution</td>
			<td>Gibco, Life Technologies</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, qualified</td>
			<td>Thermo Scientific</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutamMAXTM-I ,100x</td>
			<td>Gibco, Life Technologies</td>
			<td>35050061</td>
			<td>L-glutamine substitute</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco, Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain,suspension</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine Hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Cayman Chemical</td>
			<td>11914-1</td>
			<td>Smoothened agonist</td>
		</tr>
		<tr>
			<td>IF staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP-goat ab</td>
			<td>Rockland</td>
			<td>600-101-215</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-Arl13b mouse monoclonal ab</td>
			<td>NeuroMab</td>
			<td>75-287</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-Pax6 rabbit polyclonal ab</td>
			<td>Covance</td>
			<td>PRB-278P</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Secondary antibody mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-goat IgG</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 donkey anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-31570</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey anti-rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-31573</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62247</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CU 500 cuvette chamber</td>
			<td>Nepagene</td>
			<td>CU500</td>
			<td></td>
		</tr>
		<tr>
			<td>EPA Electroporation cuvette (2 mm gap)</td>
			<td>Nepagene</td>
			<td>EC-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies LTD</td>
			<td>31985070</td>
			<td>reduced-serum medium for transfection</td>
		</tr>
		<tr>
			<td>pEGFP-mSmo</td>
			<td>Addgene</td>
			<td>25395</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Electroporator NEPA21 TYPE II In Vitro and In Vivo Electroporation</td>
			<td>Nepagene</td>
			<td>NEPA21</td>
			<td>electroporator</td>
		</tr>
	</tbody>
</table>

efficient and cost effective electroporation method to study primary cilium-dependent signaling pathways in the granule cell precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Using the Opti-MEM (see the Table of Materials) as the universal reagent, this proposed electroporation methodology could achieve consistently high electroporation efficiency at ~80-90% (Figure 1). The electroporation efficiency of the Smo-EGFP vector was determined at DIV 2 post electroporation by quantification of the percentage of green fluorescence-positive cells in all paired box protein-6 (Pax6)-expressing GCP cells. The electroporation efficiency of DMSO- and SAG-trea...

Watch this Scientific Journal Video about Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor at JoVE.com

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

This protocol demonstrates a straightforward in vitro genetic modification method for the molecular study of the primary cilium-dependent signaling pathway in the primary granule cell precursor cultures. Due to the cost, low viability, and poor efficiency of the current transfection method, we introduce a simple, cost effective, and efficient electroporation technique for investigating cilium-dependent signaling pathway in primary GCP cultures. To begin, add 0.5 milliliters of culture medium into each well of the 24-well culture plate containing coated cover slips, and keep it warm at 37 degrees Celsius in a carbon dioxide incubator.Pipette the required number of cells into a sterile 1.5-milliliter microcentrifuge tube and spin at 200x G for five minutes at room temperature. Discard the supernatant and resuspend the cell pellet in 200 microliters of Opti-MEM. Repeat this procedure twice to ensure no residual culture medium is present in the tube.Set the parameters of electroporation as listed. Pipette the electroporation reaction gently to mix well, and use a long P200 pipette tip to transfer an exact volume of 100 microliters of the mixture into the two-millimeter gap cuvette. Once the cuvette is inside the cuvette chamber, press the omega button of the electroporator and note the impedance value by adhering to a precise volume of 100 microliters.The range of impedance value should be approximately 30 and 35. Press the start button to initiate the pulse. Record the measured current values in Joules shown on the reading frame.Remove the cuvette from the chamber. Immediately add 100 microliters of pre-warmed culture medium into the cuvette and resuspended it by pipetting up and down two to three times. Immediately transfer the cell suspension to the 24-well plate previously prepared.Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator. Observe the cells under the fluorescence microscope on the following day. In the present study, the electroporation efficiency of DMSO and SAG-treated groups appeared comparable.Immunostaining of the primary cilium marker Arl13b demonstrates that the ciliation rate of GCP at div-2 of culture in both the vehicle and SAG-treated groups. The ciliation rate illustrates the percentage of Pax6 expressing GCPs bearing a primary cilium on the cell surface at div-2 post-electroporation. The figure shows a significant increase in smoothened EGFP localization on the primary cilium axoneme of Pax6 expression GCP cells at 24 hours post-SAG treatment, indicating a profound activation of the primary cilium-dependent hedgehog signaling pathway.In order to achieve a higher electroporation efficiency, one should ensure high purity of the plasmid used, and no residue culture media is present in the plasmid electroporation mixture. This protocol should be directly applicable and beneficial for in vitro genetic modification experiments on primary cultures and cell types that are difficult to transfect, including neurons and human-induced pluripotent stem cells.

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Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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, ...