The authors wish to thank Dr. Alistair Garratt (Max Delbr&uuml;ck Center, Berlin) for helpful comments. This work was supported by the collaborative research center (SFB665) of the German Research Council (DFG).

1. Dissection procedure
Note: Experimental use of mice should follow officially approved guidelines for the care and use of laboratory animals.
<ol>
	<li>Before preparation, set up your dissecting microscope and lay out the surgical instruments needed for dissection including large and small scissors, large toothed forceps, curved forceps and four sets of Dumont No.5 dissecting forceps (two of which have inside-polished tips) (for details see the table of specific reagents and eq...

<ol>
	<li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+diO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends. Neurosci. 12 (9), 333-333 (1989).</li><li>Acebes, A., Ferrus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+features+of+axon+collaterals+and+dendrites.">Cellular and molecular features of axon collaterals and dendrites.</a> Trends. Neurosci. 23 (11), 557-557 (2000).</li><li>Ozaki, S., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+trajectories+of+sensory+axons+toward+laminar+targets+in+the+developing+mouse+spinal+cord.">Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord.</a> J. Comp. Neurol. 380 (2), 215-215 (1997).</li><li>Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+receptor+guanylyl+cyclase+Npr2+is+essential+for+sensory+axon+bifurcation+within+the+spinal+cord.">The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord.</a> J. Cell Biol. 179 (2), 331-331 (2007).</li><li>Gibson, D. A., Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+axon+branching+in+the+vertebrate+nervous+system.">Developmental regulation of axon branching in the vertebrate nervous system.</a> Development. 138 (2), 183-183 (2011).</li><li>O'Leary, D. D., Terashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+axons+branch+to+multiple+subcortical+targets+by+interstitial+axon+budding:+implications+for+target+recognition+and+&amp;quot;waiting+periods&amp;quot;.">Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and &amp;quot;waiting periods&amp;quot;.</a> Neuron. 1 (10), 901-901 (1988).</li><li>Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS. Biol. 3 (8), e272-e272 (2005).</li><li>Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+imaging+and+ultrastructural+analysis+of+individual+axon+terminals+labeled+by+iontophoretic+application+of+lipophilic+dye.">Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.</a> J. Neurosci. Methods. 93 (1), 13-13 (1999).</li><li>Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-type+natriuretic+peptide+(CNP)+is+a+bifurcation+factor+for+sensory+neurons.">C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (39), 16847-16847 (2009).</li><li>Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulate+axon+branching+by+the+cyclic+GMP+pathway+via+inhibition+of+glycogen+synthase+kinase+3+in+dorsal+root+ganglion+sensory+neurons.">Regulate axon branching by the cyclic GMP pathway via inhibition of glycogen synthase kinase 3 in dorsal root ganglion sensory neurons.</a> Journal of Neuroscience. 29 (5), 1350-1350 (2009).</li><li>Zhao, Z., Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+axonal+development+by+natriuretic+peptide+hormones.">Regulation of axonal development by natriuretic peptide hormones.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (42), 18016-18016 (2009).</li><li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+mechanisms+regulating+axonal+branching+in+vivo.">Signalling mechanisms regulating axonal branching in vivo.</a> Bioessays. , (2010).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-41 (2000).</li><li>Livet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450 (7166), 56-56 (2007).</li></ol>

The authors have nothing to disclose.

Stereotyped projection patterns comprising both types of axonal branch formation together with ease of preparation in combination with the use of fixed tissue for DiI labeling makes the embryonic spinal cord with attached DRG a favourable model to study axonal branching. The application of minute amounts of DiI using coated glass needles allows - in contrast to the bulk labeling of DRG - the visualization of small groups of DRG neurons and thereby the analysis of individual axons and their branching patterns. The describ...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Stereomicroscope Stemi DRC</td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate-buffered solution (PBS)</td>
 <td>Biochrom AG</td>
 <td>L182-50</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Merck</td>
 <td>8.18715.1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Standard surgical scissors</td>
 <td>Fine Science Tools</td>
 <td>14001-13</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Toothed standard forceps</td>
 <td>Fine Science Tools</td>
 <td>11021-14</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Extra fine iris scissors</td>
 <td>Fine Science Tools</td>
 <td>14088-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Curved forceps</td>
 <td>Fine Science Tools</td>
 <td>11003-13</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumont No.5 fine tips forceps</td>
 <td>Fine Science Tools</td>
 <td>11254-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumont No.5 mirror finish forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-23</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vannas-T&uuml;bingen spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15008-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filter paper</td>
 <td>Fisher Scientific</td>
 <td>FB59041</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sylgard 184</td>
 <td>World Precission Instruments</td>
 <td>SYLG184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100-mm Petri dishes</td>
 <td>Greiner</td>
 <td>663102</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12-ml polypropylene tube</td>
 <td>Carl Roth GmbH</td>
 <td>ECO3.1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12-well culture plate</td>
 <td>Becton Dickinson</td>
 <td>35-3043</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol </td>
 <td>Merck</td>
 <td>1.00983.2500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown micropipette puller P-97</td>
 <td>Sutter Instrument Co.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Borosilicate glass capillaries</td>
 <td>Harvard Apparatus</td>
 <td>30-0066</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DiI (1,1&prime;-Dioctadecyl-3,3,3&prime;,3&prime;-tetramethyl-indocarbocyanine perchlorate)</td>
 <td>Sigma-Aldrich</td>
 <td>468495</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope slides SuperFrost Plus</td>
 <td>Carl Roth GmbH</td>
 <td>H867.1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass cover slips</td>
 <td>Carl Roth GmbH</td>
 <td>1870.2</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

Watch this Scientific Journal Video about DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord at JoVE.com

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

dii-labeling-drg-neurons-to-study-axonal-branching-whole-mount

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Neuroscience

24.7K visualizações.  Max Delbrück Center for Molecular Medicine. As projeções estereotipadas de aferentes sensoriais para a medula espinhal de roedores oferecem um sistema de fácil acesso experimental para o estudo de ramificação axonal através do rastreamento de axônios individuais.