The collagen matrix assay has been developed and improved by the work of many different research groups during the past two to three decades. The approaches described here for dopaminergic and striatal explants greatly benefit from these studies. In addition, the authors would like to thank Asheeta Prasad for her help in setting up striatal explant cultures. Work in the lab was funded by the Human frontier Science Program Organization (Career Development Award), the Netherlands Organization of Health Research and Development (ZonMW-VIDI and ZonMW-TOP), the Europanian Union (mdDA-NeuroDev, FP7/2007-2011, Grant 222999) (to RJP), and the Netherlands Organization for Scientific Research (TopTalent; to ERES).

1. Preparation of Rat Tail Collagen
<ol>
 <li>Collect 6-10 adult rat tails (it is possible to store the tails at -20 &deg;C until use).</li>
 <li>Soak the tails in 95% ethanol overnight at room temperature (RT).</li>
</ol>
Dissection of rat tails (in tissue culture hood): 
(keep tools in 70% ethanol when not using them and make sure that all solutions, tools and glassware used throughout this procedure are sterile) 
<ol start="3">
 <li>To collect tendons from the tails, cut off the tip of the tail. Hold large end of the tail with a pair of forceps. Use another pair of forceps to hold, bend and break the tail close to the other forceps. Pull apart and the tendons will be left behind (Figure 1A,B).</li>
 <li>Collect the tendons in sterile H2O and after collecting all tendons move them to a new petridish with sterile H2O.</li>
 <li>Take 2-3 tendons and shred them using 2 pairs of forceps in a new petridish with sterile H2O. Remove non-tendon tissue such as veins (tendon tissue is shiny and reflective; Figure 1C). After removal of all non-tendon tissue, each tail yields approximately 100-150 mg of tendons.</li>
 <li>Transfer the tendons to 300 ml of sterile 3% acetic acid and stir slowly overnight at 4 &deg;C.</li>
 <li>Add an additional 200 ml of sterile 3% acetic acid and stir slowly overnight at 4 &deg;C.</li>
 <li>Centrifuge the acetic acid solution containing the tendons at ~2700 xg for 120 min at 4 &deg;C to pellet non-dissolved tendons and non-tendon tissue.</li>
 <li>During centrifugation prepare dialysis tubing:
 <ul>
 <li>cut pieces of 40 cm</li>
 <li>boil in 500 &mu;M EDTA for 5 min</li>
 <li>cool in sterile H2O</li>
 </ul>
 </li>
 <li>Tie a knot at one end of the dialysis tubing and fill the tubing with the supernatant of the centrifuged tendon solution on ice in the tissue culture hood using an automatic pipette and a 25 ml pipette. Avoid producing bubbles. Knot the other end of the tubing and store tubing on sterile aluminum foil on ice until all pieces of tubing are filled (Figure 1D).</li>
 <li>Prepare 10 l of sterile 0.1X MEM, pH 4.0, in the tissue culture hood. Add floater to pieces of tubing and add these to the pre-cooled 0.1X MEM solution in a 10 l beaker. Dialyze overnight at 4 &deg;C while slowly stirring. This dialysis removes excess acid while keeping the pH low.</li>
 <li>Replace with new pre-cooled 10 l 0.1X MEM and stir overnight at 4 &deg;C.</li>
 <li>Repeat the previous step by replacing with new pre-cooled 10 l 0.1X MEM and stir overnight at 4 &deg;C.</li>
 <li>Aliquot collagen solution into sterile 50 ml tubes. Store aliquots at 4 &deg;C. Keep 6-12 months, only handle the collagen stock in the tissue culture hood.</li>
 <li>It is important to test whether the purified collagen needs to be diluted (in 0.1x MEM) to obtain optimal results in the collagen matrix assay. Therefore, generate a collagen dilution series (undiluted collagen, 1:1, 1:2 and 1:5 diluted) and perform collagen matrix assays as described below to determine the optimal collagen dilution.</li>
</ol>
2. Dissection of the Dopaminergic Midbrain7,8
<ol>
 <li>Dissect E14.5 mouse embryos from the uterus of the mother and keep in L15 medium on ice until needed.</li>
</ol>
All subsequent dissection steps are performed in L15 medium on ice.
<ol start="2">
 <li>Dissect out the brain.</li>
 <li>Remove the telencephalon by cutting along the medial part of each telencephalic vesicle (use the telencephalic vesicles for striatal explants).</li>
 <li>Remove the meningeal sheath.</li>
 <li>Make a dorsoventral cut just rostral of the mesencephalic flexure.</li>
 <li>Make another dorsoventral cut just caudal of the mesencephalic flexure.</li>
 <li>Make a rostrocaudal cut along the dorsal midline using a microdissection knife to expose the underlying ventral midbrain tissue. Be careful not to hit the ventral midbrain tissue as it contains the dopaminergic neurons (Figure 2).</li>
 <li>Make two rostrocaudal cuts lateral and parallel to the ventral midline to remove dorsal midbrain tissue.</li>
 <li>Divide the remaining ventral midbrain tissue into explants using a microdissection knife.</li>
 <li>Store the explants in L15 medium containing 5% FBS on ice until use.</li>
</ol>
3. Dissection of the Striatum
All dissection steps are performed in L15 medium on ice.
<ol>
 <li>Use telencephalic vesicles dissected during midbrain dissection.</li>
 <li>Remove the thalamus by cutting in between the thalamus and the striatum using forceps.</li>
 <li>Make a mediolateral cut rostral to the striatum to remove rostral structures such as the olfactory bulb. Repeat this step for tissue located caudally to the striatum.</li>
 <li>Position the remaining slice to obtain a coronal view of the striatum (Figure 2).</li>
 <li>The striatum can be recognized as a slightly less dense (more transparent) part of tissue. Dissect out the striatum and cut into explants using a microdissection knife. Avoid the slightly darker tissue close to the midline, which contains migrating neurons of the lateral and medial ganglionic eminence.</li>
 <li>Store the explants in L15 medium containing 5% FBS on ice until use.</li>
</ol>
4. Assembly Collagen Matrix Assays
Preparation of the collagen (all steps on ice, use cooled pipette tips)
<ol>
 <li>Mix 860 &mu;l of diluted collagen with 100 &mu;l of 10x MEM and 40 &mu;l of 1M sodium bicarbonate (NaHCO3) and keep on ice. If at this point the collagen warms up it will solidify.</li>
 <li>Add a drop of 20 &mu;l (approximately 5 mm in diameter) of prepared collagen to a coverslip in a well of a 4-well Nunc dish and let it stand in a CO2 incubator at 37 &deg;C and 5%CO2 for 30 min (ambient O2 concentration is sufficient). During this incubation the collagen will gelatinize.</li>
</ol>
Setup co-culture in collagen gel
<ol start="3">
 <li>After the collagen has gelatinized, use a pipette with a 200 &mu;l tip to transfer a dopaminergic or striatal explant to the collagen.</li>
 <li>Move the explants in close proximity to each other using a needle. Keep them apart at a distance of approximately the diameter of one explant (~200-300 &mu;m) (Figure 2).</li>
 <li>Remove excess medium and add 20 &mu;l of prepared collagen on top of the explants. This will often cause the explants to move around. Reposition the explants using a needle as described in 4.4.</li>
 <li>Let the collagen solidify for 15 min at RT followed by 30 min at 37 &deg;C and 5% CO2.</li>
 <li>After the collagen has set, add 400 &mu;l of explant medium and grow for 2-3 days in a CO2 incubator at 37 &deg;C and 5% CO2.</li>
</ol>
5. Analysis by Immunohistochemistry and Quantification
Immunohistochemistry
<ol>
 <li>To fix explants, gently add 400 &mu;l of 8% paraformaldehyde (PFA) in PBS to 400 &mu;l of medium (thereby diluting the PFA to 4%) and let stand for 1 hour at RT.</li>
 <li>Wash 3x 15 min in PBS at RT.</li>
 <li>Incubate in blocking buffer (BB; PBS + 1% FBS + 0.1% Triton X-100) for 2 hours.</li>
 <li>Incubate overnight with primary antibody in BB at 4 &deg;C. For dopaminergic axons use anti-tyrosine hydroxylase antibodies (1:1000)7, for striatal axons anti-DARPP32 antibodies (1:500)9 and for visualizing all axons anti-&beta;III tubulin (1:3000)7.</li>
 <li>Wash 5x 1 hour in PBS at RT.</li>
 <li>Incubate with secondary antibody conjugated to the appropriate fluorophore (1:500) in BB overnight at 4 &deg;C. From this step on minimize exposure of the explants to light (e.g. cover them with aluminum foil).</li>
 <li>Wash overnight in PBS at 4 &deg;C followed by several washes during the next day.</li>
 <li>Mount explants on microscope slides using Prolong Antifade reagent mounting medium. Add a drop of ~10 &mu;l on a glass microscope slide. Take the coverslip and gently place it on the drop of mounting medium with the explant side facing down. Avoid trapping any air under de coverslip by very gently lowering it on the drop of mounting medium.</li>
</ol>
Quantification by calculating P/D ratio
<ol start="9">
 <li>Acquire digital images of the explants using an epifluorescence microscope (Figure 3).</li>
 <li>Using these images, divide each explant into quadrants to generate a proximal quadrant (i.e. part of the explant closest to the adjacent explant) and a distal quadrant (part of the explant farthest away from the adjacent explant) (Figure 2, 4).</li>
 <li>Measure the length of 20 longest neurites emerging from the explant both in the proximal and distal quadrants.</li>
 <li>Use the average length of the 20 longest neurites to calculate the P/D ratio for each individual explant. A P/D ratio &gt; 1 indicates axon attraction, while a ratio &lt; 1 indicates axon repulsion.</li>
 <li>In some cases, the dense growth of neurites prevents the assessment of individual neurite length. In these situations, quantification can be performed by measuring the distance between the edge of the explant and the leading front of axon growth in the proximal and distal quadrants.</li>
</ol>
6. Representative Images
After successful culture of dopaminergic and striatal explants a large number of axons are visible using bright field microscopy or as visualized by anti-&beta;III tubulin immunocytochemistry (not shown). A subset of these axons will be dopaminergic or striatal axons as visualized using immunocytochemistry (Figure 3). By dividing the explants into quadrants as described and determining P/D ratios, a putative axon attractive or repulsive effect can be quantified (Figure 3E).
<img alt="Figure 1" src="/files/ftp_upload/3691/3691fig1.jpg" /> 
Figure 1. Photos illustrating the preparation of collagen from rat tail tendons. A) Pulling apart the rat tail exposes the tendons. B) Tendons are visible as rope-like bundles. C) The tendons are easy to distinguish from veins by their shiny white appearance. D) After dissolving the tendons in acetic acid, the solution is transferred to dialysis tubing. To keep the dissolved collagen cold, tubes are placed on sterile aluminum foil on ice.


<img alt="Figure 2" src="/files/ftp_upload/3691/3691fig2.jpg" /> 
Figure 2. Schematic indicating the different steps of the procedure. Appropriate brain regions are dissected and cut to generate explants. A single midbrain and striatal explant are positioned in close proximity in a collagen matrix and left to grow for 48-72 hours at 37 &deg;C. Axons are visualized by fluorescence immunohistochemistry and a P/D ratio is calculated to quantify chemotropic responses.
<img alt="Figure 3" src="/files/ftp_upload/3691/3691fig3.jpg" /> 
Figure 3. Representative results showing axon growth in a collagen matrix culture. A) Midbrain explant stained with anti-tyrosine hydroxylase antibody revealing axon outgrowth. Dotted line indicates adjacent explant. B) Magnification of A showing individual neurites and growth cones (arrows). C) Striatal explant stained with anti-DARPP32. D) Magnification of C showing individual neurites. E) Example of P/D ratio quantification. Explants are divided into equal quadrants. The proximal quadrant is facing the adjacent explant while the distal quadrant is facing away from it. Scale bars indicate 100 &mu;m.

<ol>
	<li>van den Heuvel, D. M., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+connected+in+the+dopamine+system.">Getting connected in the dopamine system.</a> Prog. Neurobiol. 85, 75-93 (2008).</li><li>Lobo, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+striatonigral+and+striatopallidal+medium+spiny+neurons+past,+present,+and+future.">Molecular profiling of striatonigral and striatopallidal medium spiny neurons past, present, and future.</a> Int. Rev. Neurobiol. 89, 1-35 (2009).</li><li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+axon+guidance.">Molecular mechanisms of axon guidance.</a> Science. 298, 1959-1964 (2002).</li><li>Chilton, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+mechanisms+of+axon+guidance.">Molecules mechanisms of axon guidance.</a> Dev. Biol. 292, 13-24 (2006).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Tessier-Lavigne, M., Placzek, M., Lumsden, A. G., Dodd, J., Jessel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+guidance+of+developing+axons+in+the+mammalian+central+nervous+system.">Chemotropic guidance of developing axons in the mammalian central nervous system.</a> Nature. 336, 775-778 (1988).</li><li>Kolk, S. M., Gunput, R. A., Tran, T. S., van den Heuvel, D. M., Prasad, A. A., Hellemons, A. J., Adolfs, Y., Ginty, D. D., Kolodkin, A. L., Burbach, J. P., Smidt, M. P., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin+3F+is+a+bifunctional+guidance+cue+for+dopaminergic+axons+and+controls+their+fasciculation,+channeling,+rostral+growth,+and+intracortical+targeting.">Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their fasciculation, channeling, rostral growth, and intracortical targeting.</a> J. Neurosci. 29, 12542-12557 (2009).</li><li>Fenstermaker, A. G., Prasad, A. A., Bechara, A., Adolfs, Y., Tissir, F., Goffinet, A., Zou, Y., Pasterkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/planar+cell+polarity+signaling+controls+the+anterior-posterior+organization+of+monoaminergic+axons+in+the+brainstem.">Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem.</a> J. Neurosci. 30, 16053-16064 (2010).</li><li>Arlotta, P., Molyneaux, B. J., Jabaudon, D., Yoshida, Y., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ctip2+controls+the+differentiation+of+medium+spiny+neurons+and+the+establishment+of+the+cellular+architecture+of+the+striatum.">Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum.</a> J. Neurosci. 28, 622-632 (2008).</li><li>De Wit, J., Toonen, R. F., Verhage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+local+retention+of+secretory+vesicle+cargo+in+cortical+neurons.">Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.</a> J. Neurosci. 29, 23-37 (2009).</li><li>G&#228;hwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends Neurosci. 20, 471-477 (1997).</li></ol>

We have nothing to disclose.

The collagen matrix assay described here has been used and improved by many different labs in the past decades to investigate a variety of axon guidance molecules and neuronal systems (e.g. see references 5-8). These studies have shown that this assay is a powerful tool for studying the effects and regulation of axon guidance molecules secreted by different (intermediate) target tissues.
However, it should be noted that the collagen matrix is a substitute for the extracellular environment (EC...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>BioWhittaker</td>
 <td>14-801F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glutamine (200mM)</td>
 <td>PAA</td>
 <td>M11-004</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>VWR International</td>
 <td>441476L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>Merck</td>
 <td>444203</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Minimum Essential Media (MEM)</td>
 <td>Gibco</td>
 <td>61100-087</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neurobasal</td>
 <td>Gibco</td>
 <td>21103-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium</td>
 <td>Gibco</td>
 <td>11415-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15070-063</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Prolong Gold Antifade Reagent</td>
 <td>Invitrogen</td>
 <td>P36930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dialysis tubing</td>
 <td>Spectrum Labs</td>
 <td>132660</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rabbit anti-Tyrosine Hydroxylase</td>
 <td>Pel-Freez</td>
 <td>P40101-0</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rabbit anti-Darpp32 (H-62)</td>
 <td>Santa-Cruz</td>
 <td>Sc-11365</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse anti-&beta;III tubulin</td>
 <td>Sigma</td>
 <td>T8660</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor labeled secondary antibodies</td>
 <td>Invitrogen</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

dissection-culture-mouse-dopaminergic-striatal-explants-three

Research

JoVE Journal

Neuroscience

25.6K Views.  University Medical Center Utrecht. Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Video: Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...