Name: generation of 3-d collagen-based hydrogels to analyze axonal growth and behavior during nervous system development
Uploaded: 2019-06-25T13:00:00
Description: Watch this Scientific Journal Video about Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development at JoVE.com

The authors thank Tom Yohannan for the editorial advice and M. Segura-Feliu for the technical assistance. This work was funded by the CERCA Programme and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (SGR2017-648). This work was funded by the Spanish Ministry of Research, Innovation and University (MEICO) through BFU2015-67777-R and and RTI2018-099773-B-100, the Spanish Prion Network (Prionet Spain AGL2017-90665-REDT), and the Institute Carlos III, CIBERNED (PRY-2018-2).

All animal experiments were performed under the guidelines and protocols of the Ethical Committee for Animal Experimentation (CEEA) of the University of Barcelona, and the protocol for the use of rodents in this study was reviewed and approved by the CEEA of the University of Barcelona (CEEA approval #276/16 and 141/15).&#160;
1. Purification of Rat Tail Collagen
<ol>
	<li>Collect adult Sprague-Dawley rat tails (8-9 weeks old) after sacrificing the animal following ethical guidelines and rin...

<ol>
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M., 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=Response+of+retinal+ganglion+cell+axons+to+striped+linear+gradients+of+repellent+guidance+molecules.">Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules.</a> Journal of Neurobiology. 37 (4), 541-562 (1998).</li><li>Knoll, B., Weinl, C., Nordheim, A., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripe+assay+to+examine+axonal+guidance+and+cell+migration.">Stripe assay to examine axonal guidance and cell migration.</a> Nature Protocols. 2 (5), 1216-1224 (2007).</li><li>von Philipsborn, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+navigation+in+substrate-bound+ephrin+gradients.">Growth cone navigation in substrate-bound ephrin gradients.</a> Development. 133 (13), 2487-2495 (2006).</li><li>Chen, H., He, Z., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+mechanisms:+semaphorins+as+simultaneous+repellents+and+anti-repellents.">Axon guidance mechanisms: semaphorins as simultaneous repellents and anti-repellents.</a> Nature Neuroscience. 1 (6), 436-439 (1998).</li><li>Jessell, T. M., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development.+The+decade+of+the+developing+brain.">Development. The decade of the developing brain.</a> Current Opinion in Neurobiology. 10 (5), 599-611 (2000).</li><li>Serafini, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+netrins+define+a+family+of+axon+outgrowth-promoting+proteins+homologous+to+C.+elegans+UNC-6.">The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.</a> Cell. 78 (3), 409-424 (1994).</li><li>Charron, F., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+brain+wiring+functions+for+classical+morphogens:+a+role+as+graded+positional+cues+in+axon+guidance.">Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance.</a> Development. 132 (10), 2251-2262 (2005).</li><li>Fournier, M. F., Sauser, R., Ambrosi, D., Meister, J. 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A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trip+of+the+tip:+understanding+the+growth+cone+machinery.">The trip of the tip: understanding the growth cone machinery.</a> Nature Reviews Molecular Cell Biology. 10 (5), 332-343 (2009).</li><li>Castellani, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocols for Neuronal Cell Culture. , (2001).</li><li>Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> Journal of Neuroscience Methods. 4 (4), 329-342 (1981).</li><li>Bornstein, M. 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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=Earliest+sensory+nerve+fibres+are+guided+to+peripheral+targets+by+attractants+other+than+nerve+growth+factor.">Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.</a> Nature. 306 (5945), 786-788 (1983).</li><li>Chedotal, A., 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=Semaphorins+III+and+IV+repel+hippocampal+axons+via+two+distinct+receptors.">Semaphorins III and IV repel hippocampal axons via two distinct receptors.</a> Development. 125 (21), 4313-4323 (1998).</li><li>Kennedy, T. E., Serafini, T., de la Torre, J. 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The growth of developing axons is mainly invasive and includes ECM degradation and remodeling. Using the procedure presented here, researchers can obtain a homogenous 3-D matrix formed by the natural type I collagen in which axons (or cells) can respond to a chemical gradient secreted by genetically-modified cells as they do in vivo. Different axonal responses to gradients of attractive or inhibitory cues (protein, lipids, etc.) can be easily compared to specific control (mock transfected cells). As advantages, we must m...

Neuronal axons, ending in axonal growth cones, migrate long distances through the extracellular matrix (ECM) of the embryo over specific pathways to reach their appropriate targets. The growth cone is the distal portion of the axon and it is specialized to sense the physical and molecular environment of the cell1,2. From a molecular point of view, growth cones are guided by at least four different molecular mechanisms: contact attraction, chemoattraction, contact repulsion, and chemorepulsion triggered by different axonal guidance cues3,4,5,6. Contact-mediated processes can be partially monitored in two-dimensional (2D) cultures on micro-patterned substrates (e.g., with stripes7,8 or spots9 containing the molecules). However, axons can navigate to their target in a non-diffusive manner by sensing several attractive and repulsive molecules from guidepost cells in the environment4,5,10. Here, we describe an easy method of 3-D culture to check whether a secreted molecule induces chemorepulsive or chemoattractive effects on developing axons.
The earliest studies aimed to determine the effects of axon guidance cues used explant cultures in three-dimensional (3-D) matrices to generate gradients simulating in vivo conditions11,12. This approach, together with in vivo experiments, allowed for the identification of four major families of guidance cues: Netrins, Slits, Semaphorins, and Ephrins4,5,6. These molecular cues and other factors13 are integrated by the growing axons, triggering the dynamics of adhesion complexes and transducing mechanical forces via the cytoskeleton14,15,16. To generate molecular gradients in 3-D cultures for axonal navigation, pioneering researchers used plasma clot substrates17, which was also used for organotypic slice preparations18. However, in 1958, a new protocol to generate 3-D collagen hydrogels was reported for studying with Maximow&#180;s devices19, a culture platform, used in several studies suitable for microscopic observations20. Another pioneer study reported collagen gel as a tool to embed human fibroblasts for studying the differentiation of fibroblasts into myofibroblasts in wound healing processes21. In parallel, Lumsden and Davies applied collagen from the bovine dermis to analyze the putative effect of nerve growth factor (NGF) on the guiding of sensory nerve fibers22. With the development of new culture platforms (e.g., multi-well plates) by different companies and laboratories, collagen cultures were adapted to these new devices6,23,24,25,26. In parallel, an extract of ECM material derived from the Engelbreth-Holm-Swarm tumor cell line was made commercially available to expand these studies27.
Recently, several protocols have been developed to generate molecular gradients with putative roles in axon guidance using 3-D hydrogels (e.g., collagen, fibrin, etc.)28. Alternatively, the candidate molecule can be immobilized at different concentration in a porous matrix (e.g., NGF29) or generated by culturing in a small region of the 3-D hydrogel cell aggregates secreting the molecule to generate a radial gradient4,23,24,25,26. The last possibility will be explained in this protocol.
The procedure presented here is an easy, fast and highly reproducible method based on the analysis of axonal growth in 3-D hydrogel cultures of the embryonic mouse brain. In comparison with other methods, the protocol is well suited for non-trained researchers and can be fully developed after a short training (1-2 weeks). In this protocol, we first isolate collagen from adult rat tails to further generate 3-D matrices in which genetically-modified cell aggregates are cultured in front of the embryonic neuronal tissue. These cell aggregates form radial chemical gradients of a candidate molecule which elicits a response for the growing axons. Finally, the evaluation of the effects of the molecule on growing axons can easily be performed using a phase contrast microscopy or, alternatively, immunocytochemical methods.

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			<td height="24" style="height:24px;width:455px;">Material</td>
			<td style="width:211px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3,3&#8242;-Diaminobenzidine tetrahydrochloride 10 mg tablets (DAB)</td>
			<td>Sigma</td>
			<td>D5905</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adult Sprague-Dawley rats (8 to 9 weeks old)&#160;</td>
			<td>Criffa-Credo, Lyon, France</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Avidin-biotin-peroxidase complex (ABC)</td>
			<td>Vector Labs</td>
			<td>PK-4000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">B27 serum-free supplement 50x&#160;</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bicinchoninic acid (BCA) protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cDNA plasmid vectors</td>
			<td></td>
			<td></td>
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			<td height="23" style="height:23px;">COS1 cell lines</td>
			<td>ATTC</td>
			<td>CRL-1570</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>16325</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">D-(+)-glucose (45% solution in water) for complete Neurobasal medium</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">D-MEM (Dulbecco&#39;s Modified Eagle Medium 1x ) for COS1 culture medium</td>
			<td>Invitrogen</td>
			<td>41966-029</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Dulbecco&#8217;s phosphate buffered saline 10x (without Ca2+ and Mg2+) (D-PBS) for cultures</td>
			<td>Invitrogen</td>
			<td>14200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Ethanol&#160;</td>
			<td>merck</td>
			<td>108543</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA)</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fluorescence mounting media (e.g., Fluoromount-G or similar)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>17984-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gelatin powder&#160;</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Glacial acetic acid (Panreac, cat. no. 211008)</td>
			<td>Panreac</td>
			<td>211008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Hank&#8217;s balanced salt solution&#160;</td>
			<td>Invitrogen</td>
			<td>24020083</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Heat-inactivated foetal bovine serum&#160;</td>
			<td>Invitrogen</td>
			<td>10108-165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Heat-inactivated horse serum&#160;</td>
			<td>Invitrogen</td>
			<td>26050-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hydrogen peroxide (H2O2, 32 to 33% in water)</td>
			<td>Sigma</td>
			<td>316989</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine 200 mM solution (100x) for complete Neurobasal and COS1 medium</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Mice pregnant female (embryonic day 12.5 to 16.5; E12.5-16.5)&#160;</td>
			<td>Criffa-Credo, Lyon, France</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modified Minimum Essential Medium Eagle (MEM)</td>
			<td>Invitrogen</td>
			<td>11012-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal antibody against class III &#946;-tubulin (clone TUJ-1)</td>
			<td>Biolegend</td>
			<td>801201</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-2 supplement 100x&#160;</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium&#160;</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Merck</td>
			<td>1,040,051,000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin solution 100x&#160;</td>
			<td>Invitrogen</td>
			<td>15140-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline 10x (PBS) for immunocytochemistry</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody: biotinylated horse anti-mouse&#160;</td>
			<td>Vector Labs</td>
			<td>BA-2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-free medium (Opti-MEM)</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide&#160;</td>
			<td>Panreac</td>
			<td>162712</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate solution 7.5%&#160;</td>
			<td>Invitrogen</td>
			<td>25080-094</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile culture grade H2O&#160;</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TritonTM X-100&#160;</td>
			<td>Sigma</td>
			<td>X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizma base&#160;</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (Trypsin (0.05% (wt/vol) with EDTA (1x)</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:455px;">Equipment</td>
			<td style="width:211px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 large and 1 small curved scissors for dissection&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-ml conical centrifuge tubes&#160;</td>
			<td>Eppendorf or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-ml conical centrifuge tubes&#160;</td>
			<td>Corning or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 haemostats&#160;&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 small straight dissecting scissors</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200-ml centrifuge tubes for centrifugation</td>
			<td>Nalgene or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200-ml sterile glass conical flasks</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-litre glass beaker</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4- and 6-well culture plates&#160;</td>
			<td>Nunc</td>
			<td>176740 and 140675</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic pipette pumps and disposable 10 ml and 25 ml filter-containing sterile plastic pipettes.</td>
			<td colspan="2">Gilson, Brand, Eppendorf or similar&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic pipettes, sterile filter tips and current sterile tips&#160;</td>
			<td>Gilson, Eppendorf or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bench top microcentrifuge with angle fixed rotor&#160;</td>
			<td colspan="2">Eppendorf, Beckman Coulter or similar</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bench top refrigerated centrifuge with swing-bucket rotor (with 1.5, 15 and 50 ml tube adaptors)</td>
			<td colspan="2">Eppendorf, Beckman Coulter or similar</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture incubator at 37 &#186;C, 5% CO2 and 95% air</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis tubing cellulose membrane</td>
			<td>Sigma</td>
			<td>D9402</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis tubing closures&#160;</td>
			<td>Sigma</td>
			<td>Z37101-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable glass pipettes</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope with dark field optics&#160;</td>
			<td>Olympus SZ51 or similar&#160;</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">High-speed refrigerated Beckman Coulter centrifuge or similar with angle fixed rotor</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow hood</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large 100-mm, 60-mm and small 35-mm &#216; cell culture dishes&#160;</td>
			<td>Nunc</td>
			<td>&#160;150679 , 150288 and 150318, respectively</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic stirrer and magnetic spin bars</td>
			<td>IKA or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McIlwain tissue chopper</td>
			<td>Mickle Laboratory Engineering</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One pair of fine straight forceps and one pair of curved forceps&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades for the tissue chopper</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels (number 15 and 11)</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two pairs of fine spatulas for transferring collagen and tissue pieces</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

generation of 3-d collagen-based hydrogels to analyze axonal growth and behavior during nervous system development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we present a widely accessible methodology to study axonal growth in 3-D hydrogel collagen cultures of embryonic mouse nervous system. To this end, we isolated collagen from adult rat tails to generate 3-D matrices in which we cultured genetically-modified cell aggregates expressing Netrin-1 or Sema3E confronted with embryonic neuronal tissue (e.g., CA region of the hippocampus). These cell aggregates formed a radially distributed gradient of the candidate molecule inside the collagen matrix. Finally, to evaluate t...

Watch this Scientific Journal Video about Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development at JoVE.com

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

This method is useful to order to analyze the role of some molecules in axonal guidance and neuronal migration during neuro system development. The main advantage of this technique is that the analysis of the results is quick and simple and doesn't require complex software which would imply a lot of training for researchers. Demonstrating the procedure will be Mirian Segura-Feliu, a technician from our laboratory and myself.To begin plate 200, 000 COS-1 cells into 35 millimeter Petri dishes and incubate with complete culture medium in a cell culture incubator in order to read 70 to 80%confluency overnight. On the following day, to transfect COS-1 cells with the DNA encoding the candidate molecule using liposome based transfection method. First mix 250 microliters of serum free medium and one to 2 micrograms DNA in a 1.5 milliliter centrifuge tube and incubate at room temperature for five minutes.To prepare a liposomal tube, add 240 microliters of the serum free medium and 10 microliters of liposomal transfection reagent, and incubate at room temperature for five minutes. After incubation, add the content of the DNA tube to the lipsomal tube. Gently mix and incubate at room temperature for 15 minutes.Replace the medium on the cultured cells with 1.5 milliliters of the serum free medium. Add the DNA liposomes mixture slowly and drop wise to the cells. Incubate in the carbon dioxide cell culture incubator for three hours.After completed incubation, replace the medium with the complete culture medium and incubate overnight in the incubator. On the following day, rinse the cells with 0.1 molar Dulbecco's PBS. Add 800 microliters trypsin EDTA per each dish and incubate for five to 15 minutes in the carbon dioxide incubator.Collect detached cells with two milliliters of complete culture medium into a 15 milliliter centrifuge tube and add 10 milliliters of the same medium. Centrifuge the cells at four degrees celsius at 130 times g for five minutes. Remove the supernatant and preserve the pellet containing COS-1 cells on ice.After preparing collagen working mixture, add 100 to 150 microliters of the collagen mixture to the pellet of the transfected cells and mix gently by pipetting up and down. Spread 45 to 50 microliters of collagen cell pellet mixture onto a 35 millimeter Petri dish to form a uniform band of collagen cells, approximately one to 1.5 centimeters long. Place the dish in the incubator at 37 degrees celsius and 5%carbon dioxide until the gelation is observed.Prepare a second strip containing control cells in a second culture dish and place it in the incubator. When gelation is completed, add three to four milliliters of 37 degrees celsius warmed COS-1 complete culture medium to each dish containing the gelled collagen cell strips and place them in the incubator. Use a fine scalpel or a tissue chopper to cut the collagen cell strips to generate small square pieces, 400 to 500 micrometers in length.Transfer all the sections from the same transfection condition to a Petri dish containing three to 3.5 milliliters of neuronal culture media. Check under a dissecting microscope the quality of the pieces and place the dishes in the incubator. Use scissors to cut embryo horns on embryonic day 16.5 from the abdominal cavity of a sacrificed pregnant female rat and place them into a large Petri dish containing cold HBSSg buffer.Place the dish in the laminar flow hood and use straight forceps to extract the embryos and place them into a new dish containing cold HBBSg. With small forceps, remove the skin, and then with curved and straight forceps, carefully dissect the brain and place into a dish containing cold HBSSg. Under a dissecting microscope, using a scalpel or fine scissors, cut the brain in half along the midline to separate both the hemispheres.Then, remove the diencephalon and with fine forceps, remove meninges and blood vessels from the brain pieces. Use 100%ethanol to clean all parts of the tissue chopper, especially PTFE cutting plate and the razor blade and keep the tissue chopper in the laminar flux hood under UV illumination for 15 minutes. Transfer each tissue piece to the cutting plate of the tissue chopper.After the tissue has been chopped, transfer the pieces from the 35 millimeter Petri dishes filled with three to four milliliters of complete NCM. Using fine tungsten needles, finish the tissue dissection in complete NCM. Check the quality of the obtained slices under the dissecting microscope, making sure that the layers are clearly identifiable in the dark field optics and dissect the region of interest with tungsten needles.Keep the good quality pieces in complete NCM medium in the carbon dioxide incubator. Visual demonstration is critical in the steps related to the preparation of the co-cultures which facilitates the words to understand the process. Place several sterile four well culture plates in the laminar flow hood and prepare a collagen working mixture as previously.Add 15 to 20 microliters of the hydrogel mixture into the bottom of each well to produce a circular collagen base. Place the dishes in the incubator until complete gelation is observed. And check the quality of the gelled collagen.With a pipette, transfer a small piece of COS-1 cell aggregate onto the hydrogel base, then use a pipette to place a tissue piece on the same base, close to the piece of cells aggregate at a distance of one explant size. After preparing a new working collagen mixture on ice, gently pipette 15 to 20 microliters of this new mixture to cover the explant and the cell aggregate, making a sandwich like hydrogel culture. Use a fine tungsten needle to reorientate the explant so it faces the cell aggregate at 500 to 600 micrometers.Return the plate to the incubator until the gelation is observed and then add 0.5 milliliters of complete NCM supplemented with 2%B27 supplement and keep cultures for 36 to 48 hours in the incubator. When hippocampal axons were confronted with Netrin-1, they grew preferentially towards the source of Netrin-1, indicating that Netrin-1 acts as a chemo attractive molecule for these axons. In the control condition, or mock transfection, all axons grew radially, without any directional preference.When hippocampal axons were confronted with Sema3E secreting cells, most of them grew opposite of the cell aggregate, indicating that Sema3E acts as a chemo repulsive molecule for these axons. This schematic representation of the axonal response and quantification method shows that in control conditions, the axons were equally distributed in both proximal and distal quadrants, shown with radial outgrowth. This indicated a proximal distal or PD ratio of one.When explants showed increased number of axons in the proximal quadrant in comparison to the distal, indicating chemo attraction, the PD ratio was greater than one. When the number of axons was higher in the distal quadrant than in the proximal one, indicating chemo repulsion, the PD ratio was lesser than one. Following this method, researchers can obtain essential information about the role of some molecules during neuronal development, which allows us to establish a starting point to further explore the functions.Although this technique is useful in neuronal development as this is, it could also be applied in pharmacological screening, angiogenesis, and tissue engineering as strategies.

generation-3-d-collagen-based-hydrogels-to-analyze-axonal-growth

Research

JoVE Journal

Neuroscience

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Designing and Implementing Nervous System Simulations on LEGO Robots

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step of the method is to develop a nervous system simulation of specific reflexive behaviors of an appropriate model organism; here we use the American Lobster. Exteroceptive reflexes mediated by decussating (crossing) neural connections can explain an animal's taxis towards or away from a stimulus as described by Braitenberg and are particularly well suited for investigation using the NXT platform.1 The nervous system simulation is programmed using LabVIEW software on the LEGO Mindstorms platform. Once the nervous system is tuned properly, behavioral experiments are run on the robot and on the animal under identical environmental conditions. By controlling the sensory milieu experienced by the specimens, differences in behavioral outputs can be observed. These differences may point to specific deficiencies in the nervous system model and serve to inform the iteration of the model for the particular behavior under study. This method allows for the experimental manipulation of electronic nervous systems and serves as a way to explore neuroscience hypotheses specifically regarding the neurophysiological basis of simple innate reflexive behaviors. The LEGO Mindstorms NXT kit provides an affordable and efficient platform on which to test preliminary biomimetic robot control schemes. The approach is also well suited for the high school classroom to serve as the foundation for a hands-on inquiry-based biorobotics curriculum.

An approach to neural network modeling on the LEGO Mindstorms robotics platform is presented. The method provides a simulation tool for invertebrate neuroscience research in both the research lab and the classroom. This technique enables the investigation of biomimetic robot control principles.

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...