We want to thank Urmas Arumae for sharing a collagen protocol and Jutta Fey and Ursula Hinz for excellent technical assistance. Furthermore we want to thank Christian F. Ackermann, Ulrike Engel and the Nikon Imaging Center at the University of Heidelberg and also Joachim Kirsch for kindly help for the video shoting. The work was partially funded through the Deutsche Forschungsgemeinschaft (SFB 592).

1. Preparation of Collagen Gels
<ol>
 <li>Prepare a stock medium, containing 455 &mu;l 10x MEM, 112 &mu;l 7.5% NaHCO3, 50 &mu;l glutamine and NaOH. The concentration and the amount of the NaOH depends on the rat- tail collagen preparation (see 1.2), the final volume of the stock medium being 1,000 &mu;l.</li>
 <li>Prepare rat-tail collagen according to Ebendal (1). Store the Collagen solution at 4 &deg;C. A degradation of the collagen solution could not be observed. After preparation of a new batch, estimate the NaOH concentration needed for the stock medium (see 1.1). Use the pH-indicator of the medium for titration of the amount and concentration of NaOH. 800 &mu;l acidic rat-tail collagen have to turn a shade of red when mixed with 210 &mu;l of the stock medium. If the concentration of NaOH is too low the collagen solution will turn yellow, due to the pH indicator of the medium. Add the collagen to the medium and mix without producing bubbles. Perform these steps on ice. The polymerization occurs within 2 hr after the collagen solution and the stock medium have been mixed and the new solution is incubated at 37 &deg;C. Test this before starting an experiment. 1.3) Mix 800 &mu;l of collagen solution with 210 &mu;l of stock medium (see 1.2). Place 100 &mu;l of this solution in wells of an 8 well chamber slide. Incubate the slides at 37 &deg;C, 5% CO2 and humid conditions in a cell culture incubator. Prepare the collagen gels under a cell culture bench and sterile conditions. The collagen gelatinizes within 2 hr.</li>
</ol>
2. Dissection of Embryonic SCGs
<ol>
 <li>Harvest embryos of time pregnant mice (E16-18) from the uterus and keep them in PBS until further needed.</li>
 <li>Dissect one embryo from the amnion. Decapitate the embryo caudal of the clavicular level. Fix the head on a silicon bottom dish with "insect-needles". The ventral side of the head has to face to you and you have to see the submandibular region. The fixation enables easier preparation.</li>
 <li>Remove the skin of the submandibular region left and right and with it the sub-cutaneous connective tissue.</li>
 <li>Remove the submandibular salivary glands from their location to clear the view onto the hyoid, the infrahyoidal muscles and the sternocleidomastoid muscle.</li>
 <li>Remove these muscles carefully to clear the view onto the larynx and the trachea. Remove the trachea and the larynx. The carotid arteries are then visible on both sides on the prevertebral muscles. The SCG is located in the bifurcation of the carotid arteries and has an oval shape. Carefully remove the ganglia and place them in PBS. Remove the ganglia gently from the dissection tools if they are sticking to them. It is beneficial to use forceps as well as a needle holder mounted with an "insect needle" for the SCG removal.</li>
 <li>Once in PBS, store the ganglia at room temperature in PBS until you dissected enough ganglia. Then clean the ganglia from blood vessels and connective tissue. Perform the SCG dissection and cleaning under a dissecting microscope placed under a laminar flow bench.</li>
 <li>Finally, place the explanted ganglia onto the gelatinized collagen gels with the help of syringe needles mounted on a syringe for easier handling.</li>
 <li>Incubate the SCG explants on the gelatinized collagen in a cell culture incubator at 37 &deg;C, with 5 % CO2 and humid conditions.</li>
</ol>
3. Treatment of SCG Explants
<ol>
 <li>After 1-2 hr incubation, cover the SCG explant containing collagen with 100 &mu;l Neurobasal cell culture medium containing B27 supplement, glutamine and antibiotics. Now the wells of the chamber slides contain a volume of 200 &mu;l (100 &mu;l gel and 100 &mu;l medium). Add another 200 &mu;l of the medium, however now containing NGF (60 ng/ml) to facilitate axonal growth. Accordingly the final NGF concentration is 30 ng/ml.</li>
 <li>
 <ol class="lalpha">
 <li>To investigate the onset of SC migration, add additional factors directly at day in vitro 0 (DIV0). Dissolve the factors in the NGF containing medium in the double concentration needed (2).</li>
 <li>In order to analyze the impact on SCs, which already started migrating along axons, add additional factors at DIV3 until DIV4 (potential stop of the experiment). To this end, carefully remove 180 &mu;l of the solution, at DIV3, and add 200 &mu;l new medium containing NGF and the additional factors of interest at the double concentration (2).</li>
 </ol>
 </li>
</ol>
4. Time-lapse Imaging
Use an inverted microscope for analyses. Various objectives can be used, defining the field of view and magnification. One important aspect, however, is the working distance of the used objective, as imaging of the SCG explants is performed through the glass slide and the collagen gel. The recording frame rate of 1/10-30 minutes showed good results (2). However, this aspect has to be adjusted to the scientific question. A normal CCD camera can be used for image acquisition. For time lapse imaging a cell culture incubation chamber has to be attached to the stage of the microscope. Incubate the tissue during imaging at 37 &deg;C, with 5% CO2 and humid conditions. Start the incubation system one hour before the start of imaging. This allows the microscope parts (e.g. objective) including the chamber slide to adjust to the temperature and prevents temperature induced drifts. Define specific areas of analyses (within on explant and between different explants) with the help of the microscope software and a software-controlled motorized stage (multiposition setup) for simultaneous analyses of different applied conditions to the SCG explants. For time-lapse imaging wildtype tissue as well as tissue from transgenic animals can be used marking the SCs (s100b:GFP) (3). For imaging fluorophores a fluorescent light source has to be implemented in the micropscope setup. Use standard filters.
5. Quantification of SC migration distances
<ol>
 <li>At the end point (DIV4 for instance), fix the collagen gels with 4% PFA in the chamber slide for 3-4 hr. Consecutively wash the collagen gels 5 times for 10 min with PBS. Apply standard protocols for immunocytochemistry (2). If you are new to the preparation of SCGs, verify the identity of the ganglion as a sympathetic ganglion by immunohistochemistry against tyrosine hydroxylase (TH) a common marker for catecholaminergic cells. During immunohistochemistry (after the primary antibody), transfer the explant containing collagen gels to 24 well plates, which have a greater volume, which is beneficial for washing the gels.</li>
 <li>After immunohistochemistry, carefully place the collagen gels on glass slides. Carefully remove the remaining solution and let the collagen dry/shrink to two dimensions. This allows easy measurements of distances (2). After this, mount the samples.</li>
 <li>Finally, measure migration distances with the help of Fiji (NIH) software. No special plugins are needed to this end. To allow correct measurements in &mu;m, check the correct imaging metadata and the settings under the Fiji, image and scale. Use Fiji also for quantification of cells.</li>
</ol>

<ol>
	<li>Ebendal, T., Rush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Use of collagen gels to bioassay nerve growth factor activity. IBRO Handh, (1989).</li><li>Heermann, S., Schm&#195;cker, J., Hinz, U., Rickmann, M., Unterbarnscheidt, T., Schwab, M. H., Krieglstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuregulin+1+type+III/ErbB+signaling+is+crucial+for+Schwann+cell+colonization+of+sympathetic+axons.">Neuregulin 1 type III/ErbB signaling is crucial for Schwann cell colonization of sympathetic axons.</a> PloS One. 6 (12), e28692 (2011).</li><li>Zuo, Y., Lubischer, J. L., Kang, H., Tian, L., Mikesh, M., Marks, A., Scofield, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> The Journal of Neuroscience. The Official Journal of the Society for Neuroscience. 24 (49), 10999-11009 (2004).</li><li>Levi-Montalcini, R., Booker, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXCESSIVE+GROWTH+OF+THE+SYMPATHETIC+GANGLIA+EVOKED+BY+A+PROTEIN+ISOLATED+FROM+MOUSE+SALIVARY+GLANDS.">EXCESSIVE GROWTH OF THE SYMPATHETIC GANGLIA EVOKED BY A PROTEIN ISOLATED FROM MOUSE SALIVARY GLANDS.</a> Proceedings of the National Academy of Sciences of the United States of America. 46 (3), 373-384 (1960).</li><li>Meintanis, S., Thomaidou, D., Jessen, K. R., Mirsky, R., Matsas, 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+neuron-glia+signal+beta-neuregulin+promotes+Schwann+cell+motility+via+the+MAPK+pathway.">The neuron-glia signal beta-neuregulin promotes Schwann cell motility via the MAPK pathway.</a> Glia. 34 (1), 39-51 (2001).</li><li>Yamauchi, J., Miyamoto, Y., Chan, J. R., Tanoue, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+directly+activates+the+exchange+factor+Dock7+to+promote+Schwann+cell+migration.">ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration.</a> The Journal of cell biology. 181 (2), 351-365 (2008).</li><li>Anton, E. S., Weskamp, G., Reichardt, L. F., Matthew, 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=Nerve+growth+factor+and+its+low-affinity+receptor+promote+Schwann+cell+migration.">Nerve growth factor and its low-affinity receptor promote Schwann cell migration.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2795-2799 (1994).</li><li>Mahanthappa, N. K., Anton, E. S., Matthew, 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=Glial+growth+factor+2,+a+soluble+neuregulin,+directly+increases+Schwann+cell+motility+and+indirectly+promotes+neurite+outgrowth.">Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 16 (15), 4673-4683 (1996).</li><li>Yamauchi, J., Chan, J. R., Shooter, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+regulate+Schwann+cell+migration+by+activating+divergent+signaling+pathways+dependent+on+Rho+GTPases.">Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (23), 8774-8779 (2004).</li><li>Gumy, L. F., Bampton, E. T. W., Tolkovsky, 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=Hyperglycaemia+inhibits+Schwann+cell+proliferation+and+migration+and+restricts+regeneration+of+axons+and+Schwann+cells+from+adult+murine+DRG.">Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG.</a> Molecular and Cellular Neurosciences. 37 (2), 298-311 (2008).</li><li>Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132 (3), 487-498 (2008).</li><li>Park, D., Don, A. S., Massamiri, T., Karwa, A., Warner, B., MacDonald, J., Hemenway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+cell+death+using+an+Hsp90+ligand.">Noninvasive imaging of cell death using an Hsp90 ligand.</a> Journal of the American Chemical Society. 133 (9), 2832-2835 (2011).</li><li>Shcherbo, D., Souslova, E. A., Goedhart, J., Chepurnykh, T. V., Gaintzeva, A., Shemiakina, I. I., Gadella, T. W. J., Lukyanov, S., Chudakov, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+and+reliable+FRET/FLIM+pair+of+fluorescent+proteins.">Practical and reliable FRET/FLIM pair of fluorescent proteins.</a> BMC Biotechnology. 9, 24 (2009).</li></ol>

No conflicts of interest declared.

The development of the peripheral nervous system is an exciting process. When the development is completed, axons are ensheathed by SCs along the entire length, which can, in humans, often be over 100 cm. To this end the correct number of the required SCs has to be established during development and the SCs also have to move along extending axons to the periphery to ensure the complete axonal coverage. This holds true for myelinated but also for unmeylinated axons. In both cases all axons are in contact with SCs a...

<table border="1">
<tbody>
 <tr>
 <td>Name 			of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue 			Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>10x 			MEM</td>
 <td>Gibco</td>
 <td>21430</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium 			Bicarbonate (7.5%)</td>
 <td>Gibco</td>
 <td>25080</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td></td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>Merck</td>
 <td>109137</td>
 <td></td>
 </tr>
 <tr>
 <td>NGF</td>
 <td>Roche</td>
 <td>1058231</td>
 <td>R&amp;D#556-NG-100</td>
 </tr>
 <tr>
 <td>Neurobasal 			Medium</td>
 <td>Gibco</td>
 <td>21103</td>
 <td></td>
 </tr>
 <tr>
 <td>B27 			Supplement</td>
 <td>Gibco</td>
 <td>17504</td>
 <td></td>
 </tr>
 <tr>
 <td>antibiotics</td>
 <td>Gibco</td>
 <td>15640</td>
 <td></td>
 </tr>
 <tr>
 <td>d-PBS</td>
 <td>Gibco</td>
 <td>14040</td>
 <td></td>
 </tr>
 <tr>
 <td>insect 			needles</td>
 <td>FST</td>
 <td>26002-20</td>
 <td></td>
 </tr>
 <tr>
 <td>syringe 			needle</td>
 <td>Braun</td>
 <td></td>
 <td>BD 			# 300013</td>
 </tr>
 <tr>
 <td>8 			well chamber slide</td>
 <td>Lab 			tek</td>
 <td>177402</td>
 <td></td>
 </tr>
 </tbody>
</table>

analyzing murine schwann cell development along growing axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Axonal growth is facilitated from SCG explants upon treatment with NGF (4) (movie scheme S1 Figure 1 scheme). This process is easily visible by any inverted microscope and can be followed by time-lapse imaging (movie S2). If a scientist is new to dissecting SCG from mouse embryos we strongly recommend a validation of the technique by a simple anti- tyrosine hydroxylase (TH) immunohistochemistry. TH is a common marker for catecholaminergic neurons (in this case sympathetic neurons) ...

Watch this Scientific Journal Video about Analyzing Murine Schwann Cell Development Along Growing Axons at JoVE.com

Analyzing Murine Schwann Cell Development Along Growing Axons

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

analyzing-murine-schwann-cell-development-along-growing-axons

Research

JoVE Journal

Neuroscience

11.6K ビュー.  University of Freiburg. ここでは、SCが伸びる軸索に沿って開発することができますているシュワン細胞（SC）の遊走アッセイを記述します。

ビデオ: Analyzing Murine Schwann Cell Development Along Growing Axons

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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 ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...