We thank Denis Flipo for image processing and analysis advice, and David W. Silversides in whose laboratory the Gata4p[5kb]-GFP mouse line was generated. Research in the Pilon laboratory is funded by CIHR, NSERC, FRQS and FRQNT.

Ethics statement
Experiments involving mice were performed following Canadian Council of Animal Care guidelines for the care and manipulation of animals used in medical research. Protocols involving the manipulation of animals were approved by the institutional ethics committee of the University of Quebec in Montreal (Comit&#233; Institutionnel de Protection des Animaux; reference number 0512-R3-650-0513).
1. Preparation of Collagen Gels
Work in a sterile fashion, under a tissue culture hood.
<ol>
	<li>Prepare complete 5x DMEM (Eagle's modified essential medium) including standard antibiotics. Dissolve 3.37 g of DMEM powder and 0.925 g of NaHCO3 in 20 ml water. Sterilize by passing through a 0.22 &mu;m filter. Add 2.5 ml of sterile 100x penicillin/streptomycin and 25 ml of sterile heat-inactivated fetal bovine serum. Store at 4 &#176;C.</li>
	<li>On ice, mix 800 &#181;l of Collagen I solution (3.77 mg/ml in 0.02 N acetic acid, filter-sterilized), 600 &#181;l of complete 5x DMEM and 17 &#181;l of 1 N NaOH. Dilute with sterile water to a final volume of 3 ml. Include relevant growth factors in the mix. We typically use GDNF at 10 ng/ml to stimulate enteric NCC migration.</li>
	<li>Deposit about 480 &#181;l in each well of a single row in a 24-well plate. Avoid bubbles. The remaining rows can be used to test the effect of other growth factors on cell migration.</li>
	<li>Let the collagen polymerize at least 1 hr in a sterile incubator at 37 &#176;C.</li>
</ol>
2. Dissection of Animals16
<ol>
	<li>Set up matings and check for vaginal plugs the next morning. Day e0.5 being noon on the day the vaginal plug is found, isolate the female and wait 12 days until e12.5.</li>
	<li>Anesthetize pregnant mice with isoflurane and euthanize by CO2 inhalation.</li>
	<li>Spray the mouse with 70% ethanol. Lift the abdomen skin and open the abdomen cavity with dissecting scissors.</li>
	<li>Remove the uterus into a glass Petri dish filled with ice-cold PBS (phosphate-buffered saline). Cut the uterus transversely between individual deciduum swellings to isolate each embryo implantation site.</li>
	<li>Work on each implantation site separately in another glass Petri dish filled with ice-cold PBS. Under a dissecting microscope, use fine forceps to remove the muscle layer of the uterus.</li>
	<li>Open the visceral yolk sac and amnion to reveal the embryo. Take care when severing the blood vessels joining the embryo to the placenta/visceral yolk sac, as they are intertwined with the developing intestines.</li>
	<li>Sever the embryo&#39;s head at the neck.</li>
	<li>Insert a closed forceps in the abdominal cavity of an embryo, just above the dark red-colored liver and let the forceps open of itself (stop applying pressure) to make a transversal opening in the abdomen cavity. Pull the opened forceps down towards the posterior end of the embryo to open the abdomen completely.</li>
	<li>Grab the connective tissue behind the liver and pull the guts out of the abdomen, being careful not to break the intestines (the colon is attached to the anus).</li>
	<li>Cut the colon to free the guts from the rest of the embryo. The cut can be made at any location along the colon. Reserve the tail portion for later.</li>
	<li>Tease out the connective tissue from the cecum, then the rest of the intestines. Be careful not to wound the intestines while doing so.</li>
	<li>Cut away the liver and stomach (on the rostral end of the small intestine), as well as the mesonephros and genital ridges if some are present.</li>
	<li>Isolate the small intestine. Again, be careful not to nip the intestine. From now on, the rosrrocaudal orientation of the gut tissue can be tracked using the sharp curvature present on the rostral end. Leave the small intestine in PBS at room temperature for as short a time as possible before embedding (see step 3.3).</li>
	<li>Finally, record the number of tail somites for each embryo to accurately determine the stage of embryonic development.</li>
</ol>
3. Sectioning of Embryonic Intestines
<ol>
	<li>Before proceeding with embryo dissections, melt 1.5 g agarose in 100 ml PBS (phosphate-buffered saline) and keep at 50 &#176;C.</li>
	<li>Pour melted agarose into an embedding mold (e.g. a closed 2 ml microcentrifuge tube that has been cut lengthwise to excise about 1/4 of the tube wall). Let the agarose cool down to approximately 42-45 &#176;C, it should be only slightly warm to the touch.</li>
	<li>Embed the embryonic intestine just before the agarose solidifies (this can be evaluated with forceps tips and will occur around 36-38 &#176;C). Holding the intestine with forceps by the folded rostral end, pull it very slowly through the agarose along the length of the mold. This helps to keep the gut straight while the agarose sets. Release the tissue as soon as it begins to resist being moved. Keep track of the rostral-caudal orientation of the gut.</li>
	<li>Put the mold in a refrigerator 2-3 minutes, to ensure the agarose has completely set.</li>
	<li>Take out the agarose from the mold (by sliding it out of the opened Eppendorf tube). With a blade, take out the excess agarose at both ends, making cuts perpendicular to the intestine.</li>
	<li>Glue the rostral end of the intestine/agarose block down on the metal stage of a microtome with vibrating blade. Trim the excess agarose on the sides of the intestine/agarose block.</li>
	<li>Mount the metal stage on the vibrating microtome chamber. If necessary, adjust the angle of the stage so the intestine is as vertical as possible (hence perpendicular to the blade). Cover the specimen with ice-cold PBS. Bring the microtome blade down to a few millimeters under the buffer surface.</li>
	<li>Make 200 &mu;m vibratome transverse cuts of the caudal-most small intestine, ensuring that each agarose slice contains a full intestinal section.</li>
</ol>
4. Culture of Intestinal Explants
<ol>
	<li>Gently deposit the freshly cut intestine/agarose slices flat onto the collagen gels with forceps, placing one slice towards the middle of each well.</li>
	<li>Incubate 3 days at 37 &#176;C, in a humid 5% CO2 atmosphere, to allow migration of NCC out of the explant.</li>
	<li>Take the intestine/agarose slices off the collagen gel very gently with forceps. Take care not to damage the gel below.</li>
	<li>Avoid touching the collagen gel directly during the subsequent incubation and wash steps in order to ensure that the cell migration pattern is not disturbed. Fix with 500 &#181;l of 4% PFA (paraformaldehyde) (in PBS) per well 1 hr at room temperature.</li>
	<li>Replace the fixative with 500 &#181;l of DAPI (4',6-diamidino-2-phenylindole) solution (5 &#181;g/ml in PBS) per well and incubate 10 min at room temperature.</li>
	<li>Wash each well 3x with 500 &#181;l of PBS for 5 min.</li>
	<li>Photograph the fluorescent cells (GFP and DAPI channels) embedded in the collagen gel within each well.</li>
</ol>
5. Image Analysis
We made extensive use of ImageJ17 to process and quantify the images generated after explant culture.
<ol>
	<li>Start by imaging a micrometer slide at the same magnification as the fluorescent cell photographs. Measure the length of one micron in pixels (using the Straight Line tool).</li>
	<li>Set the scale (Analyze/Set Scale; input number of pixels/micron).</li>
	<li>For each GFP fluorescence photograph, change the image format to 8-bit grayscale (Image/Type/8-bit).</li>
	<li>Adjust intensity of the signal (Image/Adjust/Brightness/Contrast).</li>
	<li>Subtract the background noise if necessary (Process/Subtract Background; adjust the rolling ball radius).</li>
	<li>Set a threshold to highlight cells and cell clumps (Image/Adjust/Threshold), then apply a watershed to divide the clumps (Process/Binary/Watershed).</li>
	<li>Analyze particles to specify regions of interest (ROI) and generate cell number statistics (Analyze/Analyze Particles; set size minimum to exclude remaining pixels).</li>
	<li>Set measurement options to include Feret&#39;s diameter (Analyze/Set Measurements).</li>
	<li>Group ROIs and measure as a whole to determine the Feret&#39;s diameter, an indication of cell spread (Analyze/Tools/ROI Manager/More/OR, then ROI Manager/Add, and ROI Manager/Measure).</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+developmental+disorders+of+the+enteric+nervous+system.">Development and developmental disorders of the enteric nervous system.</a> Nat. Rev. Gastroenterol. Hepatol. 10 (1), 43-57 (2012).</li><li>Sasselli, V., Pachnis, V., Bursn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enteric+nervous+system.">The enteric nervous system.</a> Dev. Biol. 366 (1), 64-73 (2012).</li><li>Sanchez, M. P., Silos-Santiago, I., 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=Renal+agenesis+and+the+absence+of+enteric+neurons+in+mice+lacking+GDNF.">Renal agenesis and the absence of enteric neurons in mice lacking GDNF.</a> Nature. 382 (6586), 70-73 (1996).</li><li>Baynash, A. G., Hosoda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+endothelin-3+with+endothelin-B+receptor+is+essential+for+development+of+epidermal+melanocytes+and+enteric+neurons.">Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.</a> Cell. 79 (7), 1277-1285 (1994).</li><li>Chalazonitis, A., Pham, T. D., 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=Neurotrophin-3+is+required+for+the+survival-differentiation+of+subsets+of+developing+enteric+neurons.">Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.</a> J. Neurosci. 21 (15), 5620-5636 (2001).</li><li>Goldstein, A. M., Brewer, K. C., Doyle, A. M., Nagy, N., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+neural+crest+cell+migration+and+ganglion+formation+in+the+enteric+nervous+system.">BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system.</a> Mech. Dev. 122 (6), 821-833 (2005).</li><li>Jiang, Y., Liu, M. T., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+and+DCC+in+the+guidance+of+migrating+neural+crest-derived+cells+in+the+developing+bowel+and+pancreas.">Netrins and DCC in the guidance of migrating neural crest-derived cells in the developing bowel and pancreas.</a> Dev. Biol. 258 (2), 364-384 (2003).</li><li>Ramalho-Santos, M., Melton, D. A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signals+regulate+multiple+aspects+of+gastrointestinal+development.">Hedgehog signals regulate multiple aspects of gastrointestinal development.</a> Development. 127 (12), 2763-2772 (2000).</li><li>Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., de Graaf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+signaling+by+receptor+tyrosine+kinase+RET+for+the+directed+migration+of+enteric+nervous+system+progenitor+cells+during+mammalian+embryogenesis.">Requirement of signaling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis.</a> Development. 129 (22), 5151-5160 (2002).</li><li>Young, H. M., Hearn, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GDNF+Is+a+chemoattractant+for+enteric+neural+cells.">GDNF Is a chemoattractant for enteric neural cells.</a> Dev. Biol. 229 (2), 503-516 (2001).</li><li>Amiel, J., Sproat-Emison, E., 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=Hirschsprung+disease,+associated+syndromes+and+genetics:+a+review.">Hirschsprung disease, associated syndromes and genetics: a review.</a> J. Med. Genet. 45 (1), 1-14 (2008).</li><li>Pilon, N., Raiwet, D., Viger, R. S., Silversides, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+pre-+and+post-gastrulation+expression+of+Gata4+within+cells+of+the+inner+cell+mass+and+migratory+neural+crest+cells.">Novel pre- and post-gastrulation expression of Gata4 within cells of the inner cell mass and migratory neural crest cells.</a> Dev. Dyn. 237 (4), 1133-1143 (2008).</li><li>Nagy, N., Goldstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-3+regulates+neural+crest+cell+proliferation+and+differentiation+in+the+hindgut+enteric+nervous+system.">Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system.</a> Dev. Biol. 293 (1), 203-217 (2006).</li><li>Nagy, A., Gertsenstein, M., Vintersen, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: a laboratory manual. , 209-250 (2003).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Byth, K. 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The authors have nothing to disclose.

We show how our ex vivo explant culture technique can be used to precisely quantify enteric NCC migration potential in the presence of GDNF. Such precise quantification is greatly facilitated by using 200 &mu;m-thick vibratome gut sections instead of large pieces of approximate size, as previously described11,12,15. Indeed, this allows us to work with a reasonable number of cells in a highly reproducible setting. Of note, the uniform distribution of fluorescent enteric NCC within the caudal-most regio...

Neural crest cells (NCC) are a transient cell type unique to vertebrates that forms many derivatives during embryo development. This cell population arises at the border of the neural plate, adjacent to non-neural ectoderm1. During neurulation, bending of the neural plate places NCC along the dorsal edge of the forming neural tube. NCC then undergo an epithelial-mesenchymal transition, segregating and migrating away from the neural tube. NCC colonize various embryonic structures, including the digestive tract where they form the entire enteric nervous system (ENS), an interconnected network of neural ganglia embedded in the intestinal wall. As recently reviewed2,3, many genes have been involved in the development of this intricate structure.
Most of the ENS is derived from a small pool of NCC originating from the vagal neural tube (i.e. around the prospective hindbrain/spinal cord boundary)4. These neural progenitors reach the foregut around embryonic day (e) 9.0 in mice and then migrate caudally within the gut mesenchyme until approximately e15.0 to colonize the whole embryonic intestines. A minor subset of colonic neural progenitors is also provided by sacral NCC, which invade the posterior gut in the opposite direction up to the cecum4. Both vagal and sacral NCC require multiple migration-, proliferation-, survival- and differentiation-promoting cues to ensure complete formation of the ENS. In this regard, animal models - especially genetically modified mice - have been instrumental in the identification of several essential extracellular ligands: GDNF (glial cell-derived neurotrophic factor), Endothelin-3, Neurotrophin-3, BMPs (bone morphogenic proteins), Netrin, as well as Sonic and Indian Hedgehog (Shh and Ihh)5-10. Of these, GDNF signaling through the tyrosine kinase transmembrane receptor RET (Rearranged during transfection) is recognized as the most critical pathway for the attraction and guidance of NCC to and within the embryonic gut. GDNF is secreted by the gut mesenchyme and forms a spatiotemporally controlled rosrrocaudal gradient that is directly chemoattractive to enteric NCC, which express RET11,12.
Amongst other functions, the ENS regulates movement within the digestive tract through its interaction with smooth muscle in the intestinal wall. Absence of neural ganglia in the terminal region of the bowel results in Hirschsprung&#39;s disease: tonic contraction of the affected segment leads to blockage, upstream accumulation of digested material and massive distention of the gut and abdomen. Hirschsprung&#39;s disease occurs approximately one in 5,000 live births. The rostro-caudal migration pattern of enteric NCC is believed to be the main contributing factor to the etiology of Hirschsprung&#39;s disease. The colon, furthest from the source of migrating NCC and last portion of bowel to be colonized, is most susceptible to defects in ENS formation. In accordance with its crucial role in enteric NCC migration, disruption of GDNF/RET signaling is the main known genetic cause of Hirschsprung&#39;s disease13.
To better study NCC and ENS development, we generated a transgenic mouse line - named Gata4p[5kb]-GFP14 - in which migratory NCC are labeled with Green Fluorescent Protein (GFP). We next perfected an ex vivo cell migration assay, adapted from published work by other groups11,12,15, that now allows precise quantification of enteric NCC migration potential in the presence of various growth factors, such as GDNF.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

The following results are representative of what can be obtained with the technique described here (Figure 1). The use of growth factors (i.e. GDNF) stimulates migration of GFP-expressing enteric NCC out of the intestinal explant and into the collagen gel (Figure 2). Though some cells come out of the explant in the absence of growth factors, these are mostly not GFP-labeled and represent passive entry. It is necessary to remove the intestinal slice from the collagen gel in order...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

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Neuroscience

9.2K Views.  UQAM. We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Video: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.