Name: a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field
Uploaded: 2012-10-13T14:00:00
Description: Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

This work is funded by the Natural Sciences and Engineering Research Council of Canada (grant #249669, and #482986) and Heart and Stroke Foundation of Canada (grant #485508). The authors thank Youssef El-Hayek and Dr. Qi Wan for their assistance in developing the experimental protocols.

All procedures involving animal handling were approved by the University of Toronto Animal Care Committee in accordance with institutional guidelines (protocol no. 20009387). The following methods should be performed using sterile tools and techniques, in a laminar flow hood where applicable.
In the protocol text below, the phrase "EFH-SFM" refers to serum free media supplemented with epidermal growth factor, basic fibroblast growth factor and heparin. EFH-SFM is used when investigating the ga...

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Neurol. 227, 210-217 (2011).</li><li>Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipids,+EGF+receptors,+and+intracellular+signals+colocalize+and+are+polarized+in+epithelial+cells+moving+directionally+in+a+physiological+electric+field.">Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field.</a> The FASEB Journal. , (2002).</li><li>Fang, K., Ionides, E., Oster, G., Nuccitelli, R., Isseroff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+relocalization+and+kinase+activity+are+necessary+for+directional+migration+of+keratinocytes+in+DC+electric+fields.">Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.</a> J. Cell Sci. 112, 1967-1976 (1999).</li><li>McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Cell+Behavior+Electrically:+Current+Views+and+Future+Potential.">Controlling Cell Behavior Electrically: Current Views and Future Potential.</a> Physiol. Rev. 85, 943-978 (2005).</li><li>Morshead, C. M., Benveniste, P., Iscove, N. N., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+competence+is+a+rare+property+of+neural+stem+cells+that+may+depend+on+genetic+and+epigenetic+alterations.">Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations.</a> Nat. Med. 8, 268-273 (2002).</li><li>Babona-Pilipos, R., Droujinine, I. A., Popovic, M. R., Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Subependymal+Neural+Precursors,+but+Not+Differentiated+Cells,+Undergo+Rapid+Cathodal+Migration+in+the+Presence+of+Direct+Current+Electric+Fields.">Adult Subependymal Neural Precursors, but Not Differentiated Cells, Undergo Rapid Cathodal Migration in the Presence of Direct Current Electric Fields.</a> PLoS ONE. 6, e23808 (2011).</li><li>Deuschl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Randomized+Trial+of+Deep-Brain+Stimulation+for+Parkinson's+Disease.">A Randomized Trial of Deep-Brain Stimulation for Parkinson's Disease.</a> N. Engl. J. Med. 355, 896-908 (2006).</li><li>Stone, S. S. 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This protocol has been adapted from the well-established methods of previous studies7-9. Galvanotactic chambers can be constructed using a variety of different techniques, including the construction of a separate glass well for confinement of cell seeding, or using CO2 laser ablation for microfabrication of the central trough10,11. Some techniques may be more laborious or costly than others. We have described a simple and cost-effective assay for constructing a NPC galvanotaxis chamber us...

<table border="1">
 <tbody>
 <tr>
 <td height="44">Name of item</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Neural Precursor Cell Isolation</td>
 </tr>
 <tr>
 <td>2M NaCl</td>
 <td>Sigma</td>
 <td>S5886</td>
 <td>11.688 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>1M KCl</td>
 <td>Sigma</td>
 <td>P5405</td>
 <td>7.456 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>1M MgCl2</td>
 <td>Sigma</td>
 <td>M2393</td>
 <td>20.33 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>155 mM NaHCO3</td>
 <td>Sigma</td>
 <td>S5761</td>
 <td>1.302 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>0.5M Glucose</td>
 <td>Sigma</td>
 <td>G6152</td>
 <td>9.01 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>108 mM CaCl2 </td>
 <td>Sigma</td>
 <td>C7902</td>
 <td>1.59 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Gibco</td>
 <td>15070</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine pancreas trypsin</td>
 <td>Sigma</td>
 <td>T1005</td>
 <td></td>
 </tr>
 <tr>
 <td>Sheep testes hyaluronidase</td>
 <td>Sigma</td>
 <td>H6254</td>
 <td></td>
 </tr>
 <tr>
 <td>Kynurenic acid</td>
 <td>Sigma</td>
 <td>K3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovomucoid trypsin inhibitor</td>
 <td>Worthington</td>
 <td>LS003086</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM </td>
 <td>Invitrogen</td>
 <td>12100046</td>
 <td></td>
 </tr>
 <tr>
 <td>F12</td>
 <td>Invitrogen</td>
 <td>21700075</td>
 <td></td>
 </tr>
 <tr>
 <td>30% Glucose</td>
 <td>Sigma</td>
 <td>G6152</td>
 <td></td>
 </tr>
 <tr>
 <td>7.5% NaHCO3</td>
 <td>Sigma</td>
 <td>S5761</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td>23.83 g dissolved in 100 ml dH2O</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td></td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>Invitrogen</td>
 <td>PMG8041</td>
 <td>Reconstitute in 1 ml of hormone mix and aliquot into 20 &mu;l units.</td>
 </tr>
 <tr>
 <td>FGF</td>
 <td>Invitrogen</td>
 <td>PHG0226</td>
 <td>Reconstitute in 0.5 ml of hormone mix and aliquot into 20 &mu;l units.</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Sigma</td>
 <td>H3149</td>
 <td></td>
 </tr>
 <tr>
 <td>Apo-transferrin</td>
 <td>R&amp;D Systems</td>
 <td>3188-AT</td>
 <td>0.1 g dissolved into 4 ml dH20</td>
 </tr>
 <tr>
 <td>Putrescine</td>
 <td>Sigma</td>
 <td>P7505</td>
 <td>Dissolve 9.61 mg into Apo-transferrin solution</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma</td>
 <td>I5500</td>
 <td>Dissolve 25 mg into 0.5 ml of 0.1N HCl and add to 3.5 ml of dH20</td>
 </tr>
 <tr>
 <td>Selenium</td>
 <td>Sigma</td>
 <td>S9133</td>
 <td></td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>Sigma</td>
 <td>P6149</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Dissection Tools</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection microscope</td>
 <td>Zeiss</td>
 <td>Stemi 2000</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Galvanotaxis Chamber Preparation</td>
 </tr>
 <tr>
 <td>Square glass cover slides</td>
 <td>VWR</td>
 <td>16004</td>
 <td></td>
 </tr>
 <tr>
 <td>6N Hydrochloric Acid</td>
 <td>VWR</td>
 <td>BDH3204-1</td>
 <td></td>
 </tr>
 <tr>
 <td>High vacuum grease</td>
 <td>Dow Corning</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>60 mm Petri dishes</td>
 <td>Fisher Scientific</td>
 <td>0875713A</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-lysine</td>
 <td>Sigma</td>
 <td>P4707</td>
 <td></td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD Biosciences</td>
 <td>354234</td>
 <td>Thaw and aliquot into 150 &mu;l units</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Invitrogen</td>
 <td>10082139</td>
 <td>Only use if inducing NPC differentiation, otherwise use SFM + EFH culture media as indicated above</td>
 </tr>
 <tr>
 <td>Counting microscope</td>
 <td>Olympus</td>
 <td>CKX41</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Live Cell Time-Lapse Imaging</td>
 </tr>
 <tr>
 <td>Silver wire</td>
 <td>Alfa Aesar</td>
 <td>11434</td>
 <td></td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 <td></td>
 </tr>
 <tr>
 <td>Heat Inactivated FBS</td>
 <td>Sigma</td>
 <td>16140071</td>
 <td></td>
 </tr>
 <tr>
 <td>PVC tubing</td>
 <td>Fisher Scientific</td>
 <td>80000006</td>
 <td>3/32"ID x 5/32"OD</td>
 </tr>
 <tr>
 <td>Bleach</td>
 <td>Clorox</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>10 cc syringe</td>
 <td>BD</td>
 <td>309604</td>
 <td></td>
 </tr>
 <tr>
 <td>18 gauge needle</td>
 <td>BD</td>
 <td>305195</td>
 <td></td>
 </tr>
 <tr>
 <td>Dremel drill</td>
 <td>Dremel</td>
 <td>Model 750</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope equipped with humidified, incubated chamber</td>
 <td>Zeiss</td>
 <td>Axiovert-200M</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
Recipes
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>2M NaCl</td>
 <td>6.2 ml</td>
 </tr>
 <tr>
 <td>1M KCl</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td>1M MgCl2</td>
 <td>0.32 ml</td>
 </tr>
 <tr>
 <td>155mM NaHCO3</td>
 <td>16.9 ml</td>
 </tr>
 <tr>
 <td>1M Glucose</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>108 mM CaCl2</td>
 <td>0.09256 ml</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>Autoclaved water</td>
 <td>74 ml</td>
 </tr>
 </tbody>
</table>
Artificial cerebrospinal fluid
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume or Mass</td>
 </tr>
 <tr>
 <td>Artificial cerebrospinal fluid</td>
 <td>30 ml</td>
 </tr>
 <tr>
 <td>Bovine pancreas trypsin</td>
 <td>40 mg</td>
 </tr>
 <tr>
 <td>Sheep testes hyaluronidase</td>
 <td>22.8 mg</td>
 </tr>
 <tr>
 <td>Kynurenic acid</td>
 <td>5 mg</td>
 </tr>
 </tbody>
</table>
Trypsin Solution
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume or Mass</td>
 </tr>
 <tr>
 <td>SFM</td>
 <td>15 ml</td>
 </tr>
 <tr>
 <td>Ovomucoid trypsin inhibitor</td>
 <td>10 mg</td>
 </tr>
 </tbody>
</table>
Trypsin Inhibitor Solution
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Autoclaved water</td>
 <td>37 ml</td>
 </tr>
 <tr>
 <td>10X DMEM/F12</td>
 <td>10 ml</td>
 </tr>
 <tr>
 <td>30% Glucose</td>
 <td>2 ml</td>
 </tr>
 <tr>
 <td>7.5% NaHCO3</td>
 <td>1.5 ml</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td>Transferrin, Putrescine solution</td>
 <td>4 ml</td>
 </tr>
 <tr>
 <td>25 mg insulin solution</td>
 <td>4 ml</td>
 </tr>
 <tr>
 <td>Selenium</td>
 <td>100 &mu;l</td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>100 &mu;l</td>
 </tr>
 </tbody>
</table>
Hormone Mix (100 ml total, store at -20 &deg;C)
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Autoclaved water</td>
 <td>37.5 ml</td>
 </tr>
 <tr>
 <td>10X DMEM/F12 (3:1)</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>30% Glucose</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>7.5% NaHCO3</td>
 <td>0.75 ml</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>0.25 ml</td>
 </tr>
 <tr>
 <td>Hormone mix</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>0.5 ml</td>
 </tr>
 </tbody>
</table>
Serum Free Media EFH-SFM: add 10 &mu;l of EGF, 10 &mu;l of FGF, and 3.66 &mu;l of Heparin FBS-SFM: add 0.5 ml FBS
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>150 &mu;l</td>
 </tr>
 <tr>
 <td>SFM</td>
 <td>3.6 ml</td>
 </tr>
 </tbody>
</table>
Matrigel Solution Matrigel aliquot should be placed in a box of ice and allowed to thaw slowly over 4-5 hours to form a viscous liquid before mixing with SFM. This will ensure the formation of a smooth layer of Matrigel substrate. If not thawed slowly, the resulting substrate will contain clumps of Matrigel, possibly hindering cell migration.
<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Volume or Mass</td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>300 mg in 10 ml ddH20</td>
 </tr>
 <tr>
 <td>SFM Heat Inactivated FBS</td>
 <td>8 ml 2 ml</td>
 </tr>
 </tbody>
</table>
Matrigel Solution Mix 8 ml of SFM with 2 ml heat inactivated FBS in a 15 cc falcon tube. Mix agarose with 10 ml ddH20 in an Erlenmeyer flask, and heat in a microwave for 30 sec in 10-sec intervals, ensuring to remove the solution from the microwave after each 10-sec interval and thoroughly mix. Following the final 10-sec microwave period, mix the agarose solution with the SFM/FBS solution and store in a 57 &deg;C water bath.
</td>
 </tr>
 </tbody>
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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.

Kinematic analysis reveals that in the presence of a 250 mV/mm dcEF, undifferentiated NPCs exhibit highly directed and rapid galvanotaxis toward the cathode (Figure 5A, Movie 1). In the absence of a dcEF, random movement of the cells is observed (Figure 5B, Movie 2). At this field strength, &gt; 98% of undifferentiated NPCs migrate for the entire 6-8 hr for which they are imaged, and since dead cells do not migrate this suggests that they remain viable during this period in the abs...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

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

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 ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

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 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.

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 ...

Remotely Supervised Transcranial Direct Current Stimulation: An Update on Safety and Tolerability

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while maintaining clinical standards. The current consensus regarding the efficacy of tDCS is that multiple treatment sessions are needed to observe targeted behavioral reductions in symptom burden. However, the requirement for patients to travel to clinic daily for stimulation sessions presents a major obstacle for potential participants, due to work or family obligations or limited ability to travel. This study presents a protocol that directly overcomes these obstacles by eliminating the need to travel to clinic for daily sessions.
This is an updated protocol for remotely supervised self-administration of tDCS for daily treatment sessions paired with a program of computer-based cognitive training for use in clinical trials. Participants only need to attend clinic twice, for a baseline and study-end visit. At baseline, participants are trained and provided with a study stimulation device, and a small laptop computer. Participants then complete the remainder of their stimulation sessions at home while they are monitored via videoconferencing software.
Participants complete computerized cognitive remediation during stimulation sessions, which may serve a therapeutic role or as a &#34;placeholder&#34; for other computer-based activity. Computers are enabled for real-time monitoring and remote control by study staff.
Outcome measures that assess feasibility and tolerance are administered remotely with the aid of visual analogue scales that are presented onscreen. Following completion of all RS-tDCS sessions, participants return to clinic for a study end visit in which all study equipment is returned.
Results support the safety, feasibility, and scalability of the RS-tDCS protocol for use in clinical trials. Across 46 patients, 748 RS-tDCS sessions have been completed. This protocol serves as a model for use in future clinical trials involving tDCS.

This manuscript provides an updated remote supervision protocol that enables participation in transcranial direct current stimulation (tDCS) clinical trials while receiving treatment sessions from home. The protocol has been successfully piloted in both patients with multiple sclerosis and Parkinson&#39;s disease.

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...