Name: mechanical manipulation of neurons to control axonal development
Uploaded: 2011-04-10T14:00:00
Description: Watch this Scientific Journal Video about Mechanical Manipulation of Neurons to Control Axonal Development at JoVE.com

We gratefully acknowledge the important contributions of Dr. Robert E. Buxbaum in the development of this methodology.

1. Making glass needles.
<ol>
	<li>An adjustable micro-needle puller is used to fabricate needles with a tapered tip about 4 mm in length and that are closed solid beams. As opposed to a long flexible tip, this short 4 mm length limits vibrations of the needle tip during experiments. At the proximal region of the 4 mm fiber, the needle tapers rapidly from the diameter of the glass tubing to 15 &mu;m within 1 mm, while the distal-most 1 mm of the fiber is 2.5 &mu;m in diameter. We use R-6 glass capillary tube, OD 0.9 ...

<ol>
	<li>Zheng, J., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+growth+cone+adhesion+to+culture+surfaces+by+micromanipulation.">Measurements of growth cone adhesion to culture surfaces by micromanipulation.</a> J Cell Biol. 127, 2049-2060 (1994).</li><li>Chada, S., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+neurite+outgrowth+from+chick+brain+neurons.">Cytomechanics of neurite outgrowth from chick brain neurons.</a> J Cell Sci. 110, 1179-1186 (1997).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E., Haynes, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neuron in Tissue Culture. , 105-119 (1999).</li><li>Lamoureux, P., Altun-Gultekin, Z. F., Lin, C., Wagner, J. A., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+is+required+for+growth+cone+function+but+not+neurite+assembly.">Rac is required for growth cone function but not neurite assembly.</a> J Cell Sci. 110, 635-641 (1997).</li><li>Lamoureux, P., Ruthel, G., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+can+specify+axonal+fate+in+hippocampal+neurons.">Mechanical tension can specify axonal fate in hippocampal neurons.</a> J Cell Biol. 159, 499-508 (2002).</li><li>Lamoureux, P., Heidemann, S. R., Martzke, N. R., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+elongation+within+and+along+the+axon.">Growth and elongation within and along the axon.</a> Dev Neurobiol. 70, 135-149 (2010).</li><li>Dennerll, T. J., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytomechanics+of+axonal+elongation+and+retraction.">The cytomechanics of axonal elongation and retraction.</a> J Cell Biol. 109, 3073-3083 (1989).</li><li>Heidemann, S. R., Kaech, S., Buxbaum, R. E., Matus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observations+of+the+mechanical+behaviors+of+the+cytoskeleton+in+living+fibroblasts.">Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.</a> J Cell Biol. 145, 109-122 (1999).</li><li>Yoneda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Exerted+by+a+Single+Cilium+of+Mytilus-Edulis+.1.">Force Exerted by a Single Cilium of Mytilus-Edulis .1.</a> Journal of Experimental Biology. 37, (1960).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Tension+Produced+by+Nerve-Cells+in+Tissue-Culture.">Mechanical Tension Produced by Nerve-Cells in Tissue-Culture.</a> Journal of Cell Science. 37, 391-410 (1979).</li><li>Pfister, B. J., Iwata, A., Meaney, D. F., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Fass, J. N., Odde, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+force-dependent+neurite+elicitation+via+anti-beta1+integrin+antibody-coated+magnetic+beads.">Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.</a> Biophys J. 85, 623-636 (2003).</li><li>Yang, S., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Force+Sensors+and+Their+Applications+in+the+Study+of+Cell+Mechanical+Response.">Microfabricated Force Sensors and Their Applications in the Study of Cell Mechanical Response.</a> Exp Mech. 49, 135-151 (2009).</li><li>Bernal, R., Melo, F., Pullarkat, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drag+Force+as+a+Tool+to+Test+the+Active+Mechanical+Response+of+PC12+Neurites.">Drag Force as a Tool to Test the Active Mechanical Response of PC12 Neurites.</a> Biophysical Journal. 98, 515-523 (2010).</li><li>Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+growth+cones+pull.">Direct evidence that growth cones pull.</a> Nature. 340, 159-162 (1989).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E. <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+behavior+and+production+of+traction+force.">Growth cone behavior and production of traction force.</a> J Cell Biol. 111, 1949-1957 (1990).</li><li>O'Toole, M., Lamoureux, P., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Heidemann, S. R., Lamoureux, P., Ngo, K., Reynolds, M., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-dish+incubator+for+live+cell+imaging+with+an+inverted+microscope.">Open-dish incubator for live cell imaging with an inverted microscope.</a> Biotechniques. 35, 708-708 (2003).</li></ol>

Techniques to apply and measure cellular forces have a long history9. Our method was originally motivated by the work of Dennis Bray, who used glass needles similar to ours to 'tow' neurons at a constant rate using a motorized hydraulic device10. There are many alternative means of applying forces to cells which include: stepper motors11, magnetic beads12, microfabricated beams13 and fluid flows14. The latter are similar to our approach in that the cellula...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>R-6 cap. Tube</td>
<td>&nbsp;</td>
<td>Drummond Scientific Co., Broomall, PA, USA</td>
<td>9-000-3111</td>
<td>R-6 glass OD 0.9mm, ID 0.6 mm, 8"</td>
</tr>
<tr>
<td>BB-CH puller</td>
<td>&nbsp;</td>
<td>Mecanex S.A., Geneva, Switzerland</td>
<td>BB-CH puller</td>
<td>Use Mode 4 Alt by CP=100, PP=10, SP1=1000, SP2=1000</td>
</tr>
<tr>
<td>0.001" Chromel wire</td>
<td>&nbsp;</td>
<td>Omega Engineering, Stamford, CT, USA</td>
<td>SPCH-001-50</td>
<td>unsheathed, themocouple wire, 50ft spool now called Chromega</td>
</tr>
<tr>
<td>0.003" Constatan wire</td>
<td>&nbsp;</td>
<td>Omega Engineering, Stamford, CT, USA</td>
<td>SPCI-003-50</td>
<td>unsheathed, themocouple wire, 50 ft spool</td>
</tr>
<tr>
<td>fine forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools, USA</td>
<td>91150-20</td>
<td>Dumont Inox #5</td>
</tr>
<tr>
<td>universal microscope boom stand</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>76135 or 90430</td>
<td>most brands or types of boom stand will work for this use</td>
</tr>
<tr>
<td>mechanical micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige </td>
<td>M-152</td>
<td>three-axis direct-drive coarse micromanipulator</td>
</tr>
<tr>
<td>hydraulic micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige </td>
<td>MO-203</td>
<td>now available as MMO-203, three movable axis type</td>
</tr>
<tr>
<td>needle holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520145</td>
<td>set of 3</td>
</tr>
<tr>
<td>single instrument holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520142</td>
<td>&nbsp;</td>
<tr>
<td>double instrument holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520143</td>
<td>&nbsp;</td>
<tr>
<td>mechanical micromanipulator</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>39430001</td>
<td>post mount,1 prob holder, RH Model 430001</td>
</tr>
<tr>
<td>joystick mech. micromanipulator</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520137</td>
<td>&nbsp;</td>
<tr>
<td>Leica DM IRB</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>&nbsp;</td>
<td>inverted microscope</td>
</tr>
<tr>
<td>Vibraplane isolation table</td>
<td>&nbsp;</td>
<td>Kinetic System, Boston, MA, USA</td>
<td>1200 series</td>
<td>ours is model 1201-02-12</td>
</tr>
<tr>
<td>Ringcubator</td>
<td>&nbsp;</td>
<td>self manufactured see reference 19</td>
<td>&nbsp;</td>
<td>reference 19, requires updated controller listed below</td>
</tr>
<tr>
<td>programable temperature controller</td>
<td>&nbsp;</td>
<td>Instrumart.com</td>
<td>Fuji Electric PXR3</td>
<td>replaces the retired PXV3 temperature controller</td>
</tr>
<tr>
<td>Nikon Diaphot TMD</td>
<td>&nbsp;</td>
<td>Nikon Instruments, Inc.</td>
<td>&nbsp;</td>
<td>inverted microscope, circa 1980</td>
</tr>
<tr>
<td>Nikon SMZ-10 binocular dissecting </td>
<td>&nbsp;</td>
<td>Nikon Instruments, Inc.</td>
<td>&nbsp;</td>
<td>other dissecting microscopes will work</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Mechanical Manipulation of Neurons to Control Axonal Development at JoVE.com

Mechanical Manipulation of Neurons to Control Axonal Development

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 ...

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