Name: the olfactory system as a model to study axonal growth patterns and morphology in vivo
Uploaded: 2014-10-30T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo at JoVE.com

This work was supported by DFG Schwerpunktprogramm 1392 (project MA 4113/2-2), cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (project B1-9), and the German Ministry of Research and Education (BMBF; project 1364480).

NOTE: Animal handling and experiments were performed as approved by the G&#246;ttingen University Committee for Ethics in Animal Experimentation.
1. Preparation of Instruments and Pipette Fabrication
<ol>
	<li>Ensure that the electroporation setup consists of a stereomicroscope with large working distance and is equipped with fluorescent illumination and filter sets for the used dye.</li>
	<li>For electroporation use either a dedicated single cell electroporator or a generic square pulse generator attached to an oscilloscope. Connect the electroporator outputs to a pipette holder and a bath electrode. Connect the positive terminal of the pulse generator to the micropipette holder and the negative terminal to the bath electrode. Ensure both the pipette holder and the bath electrode contain silver wires coated with a thin layer of silver chloride.</li>
	<li>Mount the pipette holder on a micromanipulator to allow exact positioning.</li>
	<li>Fabricate electroporation micropipettes from borosilicate glass capillaries with internal filament.</li>
	<li>Use a horizontal micropipette puller and apply a modified protocol for the fabrication of pipettes for patch clamp experiments as described by Bestman et al.13.</li>
	<li>Adapt parameters to produce a longer shank and a smaller tip opening resulting in a higher pipette resistance of around 15-20 MOhm for single cell electroporation. The pipette resistance should be lower, e.g., 3-4 MOhm, for labeling of groups of cells.</li>
	<li>Measure the pipette resistance either directly with a dedicated single-cell electroporator or calculate it with Ohm&#8217;s Law after measuring current flow with an oscilloscope after application of a defined voltage pulse.</li>
</ol>
2. Preparation of Electroporation Solution
<ol>
	<li>Dissolve fluorophore-coupled dextran in frog ringer (98 mM NaCl, 2 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose, 5 mM Na-pyruvate, 10 mM HEPES, adjusted to pH 7.8, osmolarity was 230 mOsmol/l) at a concentration of 3 mM. The cells may not be so brightly labeled if lower dye concentrations are used. Prepare a large volume stock solution and divide it into small aliquots. Freeze them for storage (stable for months). 
	NOTE: Dextrans are available in different sizes and a variety of emission/excitation spectra (e.g., Alexa 488-dextran 10kD, Alexa 546-dextran 10kD, Alexa 568-dextran 10kD, Alexa 594-dextran 10kD, TMR-dextran 3kD).</li>
	<li>Backfill the micropipette with an elongated pipette tip with a small volume of dextran solution (1-5 &#181;l). Carefully flick the micropipette with the finger to remove residual air bubbles from the pipette tip.</li>
	<li>Mount the micropipette in the pipette holder. Make sure that the silver wire inside the pipette is in contact with the dye solution.</li>
</ol>
3. Selection of Larval X. laevis
<ol>
	<li>Use albino larvae of X. laevis for the experiments. Wild type animals possess pigment-filled melanophores that show autofluorescence emission during confocal/multiphoton imaging and are therefore not suitable for the described experiments.</li>
	<li>Stage premetamorphotic tadpoles after Nieuwkoop and Faber14. Select tadpoles of stages 45-53 for the experiments.</li>
</ol>
4. Electroporation of Fluorophore-coupled Dextrans
<ol>
	<li>Place a small piece of tissue in a petri dish and cover it with a small volume of water containing 0.02% tricaine (Ethyl 3-aminobenzoate methanesulfonate, adjusted to pH 7).</li>
	<li>Anesthetize the tadpoles in water containing 0.02% tricaine. After some minutes the animals cease moving. Confirm proper anesthesia by touching tadpoles. They should be non-responsive.</li>
	<li>Carefully transfer the tadpole from the anesthetic to the tissue covered petri dish.</li>
	<li>Make sure the bath electrode closes the electroporation circuit. Ensure that the electrode is in contact with the wet tissue; a direct contact to the tadpole is not necessary.</li>
	<li>Position the micropipette tip close to the olfactory organ using the micromanipulator.</li>
	<li>Penetrate the skin covering the olfactory organ with the pipette tip and cautiously advance the tip into the centrally located sensory neuron layer of the main olfactory epithelium or vomeronasal epithelium.</li>
	<li>Trigger positive square voltage pulses to transfer dye into sensory neurons. Apply a single voltage pulse (e.g., 25 V, pulse length 25 msec) or trains of multiple pulses (e.g., 50 V, pulse length 300 &#181;sec, 400 msec train duration at 200 Hz). 
	NOTE: Determine optimal voltage pulse parameters for the desired extend of labeling. Reduce voltage pulse amplitude, duration and number of repetitions to lower the number of labeled cells. Apply higher voltage pulse amplitude, duration and number of pulses for a more widespread labeling.</li>
	<li>Visualize successful dye extrusion and electroporation by triggered pulses using&#160;fluorescent illumination of the stereomicroscope. The dye spreads quickly into cell body and dendrite after successful electroporation.</li>
	<li>Repeat steps 4.5-4.9 for the second olfactory organ of the tadpole.</li>
	<li>Transfer the tadpole into a beaker filled with fresh water for recovery. After ca. 5 min the tadpoles wake from anesthesia and start normal swimming movements.</li>
	<li>After 24 hr the electroporated dye spreads in the sensory neurons and eventually reaches the olfactory bulb via axonal transport.</li>
</ol>
5. Mounting Animals for In Vivo Visualization of Cells and Axonal Processes
<ol>
	<li>Anesthetize the tadpoles in water containing 0.02% tricaine.</li>
	<li>Carefully transfer tadpoles into an imaging chamber, e.g., a small silicon rubber covered petri dish with a tadpole-sized recess.</li>
	<li>Cut a small rectangle into a stripe of Parafilm. Cover the tadpole with the Parafilm, leaving the anterior telencephalon exposed through the cut-out window. Fix the Parafilm with needles on the dish without injuring the tadpole.</li>
	<li>Make sure the tadpole is submerged in sufficient water containing 0.02% tricaine.</li>
	<li>Mount the imaging chamber on the stage of an upright multiphoton microscope or confocal microscope.</li>
	<li>Acquire a three dimensional stack of images of the olfactory bulb. Ensure that the imaging procedure is as short as possible and does not exceed 10-15 min.</li>
	<li>Return the tadpole into normal water in a separate tank and prevent exposure to light. After 5 min, the tadpole wakes from anesthesia.</li>
	<li>Repeat steps 5.1-5.7 after specified time intervals, e.g., every day.</li>
</ol>
6. Mounting Animals for Ex Vivo Visualization of Cells and Axonal Processes
<ol>
	<li>Alternatively to section 5 of the protocol, use an excised brain preparation to visualize the labeled sensory neurons.</li>
	<li>Anesthetize and kill the tadpole by transection of the brain at the transition to the spinal cord. Excise a block of tissue containing the olfactory organs, the olfactory nerves and the anterior telencephalon.</li>
	<li>Transfer the tissue block to frog ringer solution and remove connective tissue with fine scissors to expose the ventral side of the brain.</li>
	<li>Transfer the tissue block to an imaging chamber and fix it with a platinum frame stringed with nylon threads.</li>
	<li>Mount the imaging chamber on the stage of a confocal/multiphoton microscope.</li>
	<li>Acquire a three dimensional stack of images of the olfactory bulb.</li>
</ol>
7. Image Processing and Data Evaluation
<ol>
	<li>Optimize and apply non-local means of filtering to improve image quality and visualization of neuronal structures as described by Coup&#233;, P. et al. 15. 
	NOTE: Data from imaging experiments in deeper tissue layers of live specimens are often noisy.</li>
	<li>Create maximum intensity projections of the image stacks for an overview.</li>
	<li>For time-lapse imaging experiments screen data sets for single unambiguously identifiable axons of sensory neurons.</li>
	<li>Reconstruct cellular morphology via software-assisted tracing of neuronal processes as described by Peng et al.16.</li>
</ol>

<ol>
	<li>Huard, J. M., Youngentob, S. L., Goldstein, B. J., Luskin, M. B., Schwob, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+olfactory+epithelium+contains+multipotent+progenitors+that+give+rise+to+neurons+and+non-neural+cells.">Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells.</a> J. Comp. Neurol. 400 (4), 486-4810 (1998).</li><li>Schwob, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regeneration+and+the+peripheral+olfactory+system.">Neural regeneration and the peripheral olfactory system.</a> Anat. Rec. 269 (1), 33-49 (2002).</li><li>Leung, C. T., Coulombe, P. A., Reed, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+olfactory+neural+stem+cells+to+tissue+maintenance+and+regeneration.">Contribution of olfactory neural stem cells to tissue maintenance and regeneration.</a> Nat. Neurosci. 10 (6), 72-726 (2007).</li><li>Lodovichi, C., Belluscio, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptors+in+the+formation+of+the+olfactory+bulb+circuitry.">Odorant receptors in the formation of the olfactory bulb circuitry.</a> Physiology (Bethesda. 27 (4), 212-2110 (2012).</li><li>Mori, K., Sakano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+is+the+olfactory+map+formed+and+interpreted+in+the+mammalian+brain).">How is the olfactory map formed and interpreted in the mammalian brain).</a> Annu. Rev. Neurosci. 34, 467-499 (2011).</li><li>De Vry, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+the+central+nervous+system:+a+non-viral+approach+for+targeted+gene+delivery.">In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery.</a> Prog. Neurobiol. 92 (3), 227-244 (2010).</li><li>Hovis, K. R., Padmanabhan, K., Urban, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+in+vitro+electroporation+allows+visualization,+recording,+and+calcium+imaging+of+local+neuronal+circuits.">A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits.</a> J. Neurosci. Methods. 191 (1), 1-10 (2010).</li><li>Chen, C., Smye, S. W., Robinson, M. P., Evans, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electroporation+theories:+a+review.">Membrane electroporation theories: a review.</a> Med. Biol. Eng. Comput. 44 (1-2), 5-14 (2006).</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70 (4-5), 148-154 (2002).</li><li>Hewapathirane, D. S., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation+in+vivo+within+the+intact+developing+brain.">Single cell electroporation in vivo within the intact developing brain.</a> J. Vis. Exp. (17), (2008).</li><li>Gliem, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimodal+processing+of+olfactory+information+in+an+amphibian+nose:+odor+responses+segregate+into+a+medial+and+a+lateral+stream.">Bimodal processing of olfactory information in an amphibian nose: odor responses segregate into a medial and a lateral stream.</a> Cell. Mol. Life Sci. 70 (11), 1965-1984 (2013).</li><li>Hassenkl&#246;ver, T., Manzini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+wiring+logic+in+amphibians+challenges+the+basic+assumptions+of+the+unbranched+axon+concept.">Olfactory wiring logic in amphibians challenges the basic assumptions of the unbranched axon concept.</a> J. Neurosci. 33 (44), 17252-1710 (2013).</li><li>Bestman, J. E., Ewald, R. C., Chiu, S. -. L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+single-cell+electroporation+for+transfer+of+DNA+and+macromolecules.">In vivo single-cell electroporation for transfer of DNA and macromolecules.</a> Nat. Protoc. 1 (3), 1272-1210 (2006).</li><li>Nieuwkoop, P. D., Faber, J., Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis. , (1994).</li><li>Coup&#233;, P., Munz, M., Manj&#243;n, J. V., Ruthazer, E. S., Collins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CANDLE+for+a+deeper+in+vivo+insight.">A CANDLE for a deeper in vivo insight.</a> Med. Image Anal. 16 (4), 849-864 (2012).</li><li>Peng, H., Ruan, Z., Long, F., Simpson, J. H., Myers, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nat. Biotechnol. 28 (4), 348-353 (2010).</li><li>Falk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+cDNA/Morpholinos+to+targeted+areas+of+embryonic+CNS+in+Xenopus.">Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.</a> BMC Dev. Biol. 7 (107), (2007).</li><li>Nezlin, L. P., Schild, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+olfactory+sensory+neurons+project+into+more+than+one+glomerulus+in+Xenopus+laevis+tadpole+olfactory+bulb.">Individual olfactory sensory neurons project into more than one glomerulus in Xenopus laevis tadpole olfactory bulb.</a> J. Comp. Neurol. 481 (3), 233-239 (2005).</li><li>Nagayama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+simultaneous+tracing+and+Ca2++imaging+of+local+neuronal+circuits.">In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits.</a> Neuron. 53 (6), 789-803 (2007).</li></ol>

The authors have nothing to disclose.

The experimental procedure described here allows labeling sensory neurons of the olfactory organ of larval X. laevis by electroporation of fluorophore-coupled dextrans and subsequent visualization of sensory axon growth in the living animal. By varying the parameters of the in vivo electroporation it is possible to control the number of labeled sensory neurons. It is thereby possible to label large groups of neurons of a sensory epithelium, very few or even single cells.
To ensure the desired extend of neuronal labeling it is important to be particularly cautious about the micropipette characteristics and the electroporation pulses. Higher pipette resistances and reduction of voltage pulse amplitude, duration and number of repetitions can reduce the amount of labeled cells, whereas decreasing pipette resistances and higher voltage pulse amplitude, duration and number of pulses can lead to more widespread labeling. The use of fluorescent dextrans for electroporation provides immediate visual feedback if the applied settings are appropriate. Be careful that using parameters for amplitude, duration and number of pulses that exceed the values provided in the protocol can potentially lead to cell damage or even cell death17. Clogged or broken tips of micropipettes can also hinder successful electroporation.
In vivo electroporation in the olfactory organ of X. laevis is limited to larval stages since the skin of postmetamorphotic frogs is tougher and cannot be easily penetrated with a micropipette. The in vivo visualization of neuronal processes can be hindered by scattering of excitation/emission light in deeper brain areas or by blood vessels. This problem becomes especially apparent in higher larval stages due to a larger brain and can lead to noisy signals making the clear identification of fine axonal processes more difficult.
The presented protocol permits to visualize sensory neurons in the intact olfactory system without dissecting the animal, damaging the cells during labeling, preparing tissue slices or fixing the tissue as necessary for alternative methods, like labeling in whole-cell patch-clamp experiments18. When combining the labeling of few or single sensory neurons with in vivo time lapse imaging, it is possible to visualize the glomerular connections of single mature sensory neurons over long time intervals. This way it is also possible to monitor the development of the axonal projection patterns of immature sensory neurons over several weeks. This latter option is particularly interesting as it allows monitoring the growth patterns of single axons in the living animal. This opens the possibility to investigate cellular and molecular mechanisms that control axon guidance and pathfinding. Several factors, including odorant receptor expression, various axon guidance molecules and odorant-induced/spontaneous activity of sensory neurons have been shown to regulate the target finding of sensory neuron&#160;axons4,5.
The application of the protocol is not restricted to olfactory sensory neurons, but can also be applied to study other cell types, e.g., stem/progenitor cells of the neurogenic zones of the developing brain or mitral cells of the olfactory bulb. In addition the demonstrated technique can also be used in combination with calcium sensitive dextrans or injected membrane-permeable calcium dyes to get functional information about the labeled neuron and/or the connected circuitry7,19. The availability of a wide range of fluorophores coupled to dextrans permits labeling of multiple individual cells or populations with different colors. Also plasmid DNA solution, for example encoding for fluorescent proteins, is suitable for electroporation and can further enhance the versatility and usefulness of the technique6. The protocol can further be enhanced to allow the combined electroporation of dextrans and DNA or charged morpholinos to manipulate gene expression13,17.
The described method certainly represents a new tool to investigate the complex and still not fully understood processes that regulate axonal guidance in the vertebrate olfactory system.

The lifelong turnover of sensory neurons distinguishes the olfactory system from many other sensory and neuronal systems1,2. Newly formed sensory neurons are continuously generated in the basal portion of the olfactory epithelium3 and extend their axons into the olfactory bulb, the first relay station of the olfactory system4. However, the cellular and molecular mechanisms controlling the formation and maintenance of the olfactory map are far from being fully understood4,5.
Here, we describe a protocol for labeling sensory neurons of the olfactory organ of larval X. laevis by in vivo electroporation of fluorophore-coupled dextrans. The presented protocol allows visualization of axonal morphology and connectivity, track axonal development over time and study mechanisms regulating axonal wiring and guidance.
Electroporation is a well established method to introduce charged macromolecules, like dextran-coupled dyes and DNA, into cells6,7. The cell membrane is permeabilized by application of short voltage pulses and the molecules are electrophoretically delivered into the cytosol8. Spatially restricted electroporation using a micropipette permits selective labeling of cells including neurons and has been applied in various neuronal systems including the visual system of X. laevis9,10.
We show how the electroporated animals can be used to study axonal growth patterns and morphology in living animals using confocal laser-scanning or multiphoton microscopy. The described procedure allows identifying the coarse topology of axonal projections of sensory neurons of the main and accessory olfactory system11,12. Using in vivo time lapse imaging, it is also suitable to supervise the glomerular connections of single mature sensory neurons, and to monitor the evolution of the axonal projection patterns of immature sensory neurons12. The described protocol can be applied to investigate the structure and formation of olfactory circuits in the intact animal and can be adapted to other cell types within the olfactory and other neuronal systems.

<table><tbody><tr><td>SZX16</td><td>Olympus</td><td></td><td>Stereomicroscope with fluorescent illumination</td></tr><tr><td>Axon Axoporator 800A</td><td>Molecular Devices</td><td></td><td>Single cell electroporator</td></tr><tr><td>ELP-01D</td><td>npi electronic</td><td></td><td>Electroporator</td></tr><tr><td>MMJ</td><td>M&amp;auml;rzh&amp;auml;user Wetzlar</td><td></td><td>Manual micromanipulator</td></tr><tr><td>P-1000</td><td>Sutter</td><td></td><td>Horizontal micropipette puller</td></tr><tr><td>G150F-4</td><td>Warner Instruments</td><td></td><td>Glass capillaries for electroporation pipette fabrication; internal filament makes backfilling easier</td></tr><tr><td>Alexa 488-dextran 10 kD</td><td>Life Technologies</td><td>D22910</td><td></td></tr><tr><td>Alexa 546-dextran 10 kD</td><td>Life Technologies</td><td>D22911</td><td></td></tr><tr><td>Alexa 568-dextran 10 kD</td><td>Life Technologies</td><td>D22912</td><td></td></tr><tr><td>Alexa 594-dextran 10 kD</td><td>Life Technologies</td><td>D22913</td><td></td></tr><tr><td>TMR-dextran 3 kD (micro-Ruby)</td><td>Life Technologies</td><td>D7162</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td><td>Sigma-Aldrich</td><td>E10521</td><td>Anesthetic; use gloves</td></tr><tr><td>A1-MP</td><td>Nikon</td><td></td><td>Multiphoton microscope</td></tr><tr><td>LSM 780</td><td>Zeiss</td><td></td><td>Confocal microscope</td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td>Alternative software for neuronal tracing</td></tr></tbody></table>

the olfactory system as a model to study axonal growth patterns and morphology in vivo

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

The described protocol can be successfully applied for electroporation and in vivo visualization of axonal processes of sensory neurons of the olfactory system of anesthetized X. laevis, using confocal laser-scanning or multiphoton microscopy (Figure 1).
Electroporation allows fluorophore-coupled dextrans to enter and to rapidly spread inside cells of the olfactory organ. It is helpful to apply fluorescent illumination to verify successful labeling after triggering of voltage pulses. Depending on the electroporation parameters, e.g., pipette resistance, groups of cells (Figure 2A, B) or single cells (Figure 2C) of the olfactory organ are labeled. Axons of labeled sensory neurons can be visualized in the olfactory nerve (Figure 2D) and axonal processes can be observed also in the olfactory bulb, usually 24 hr after successful electroporation (Figure 3). Electroporation of groups of sensory neurons allows visualizing the coarse wiring patterns in the olfactory bulb (Figure 3A). Single cell electroporation can be applied to investigate individual axonal projection patterns, axonal bifurcations and connectivity to glomerular structures in the olfactory bulb (Figure 3C-E).
Transparent albino tadpoles of X. laevis permit in vivo confocal or multiphoton imaging of labeled sensory neurons in the intact brain of anesthetized tadpole. The development of axonal growth patterns can be tracked over several days/weeks by repeated in vivo visualization of the same labeled sensory neuron (Figure 4). Depending on the maturity of the sensory neuron at the time point of electroporation the axon pattern in the olfactory bulb can vary considerably. Mature neurons already possess elaborate axonal branches that feature tufted areas connected to glomeruli. The axonal growth pattern of mature neurons is rather stable, but elongation of axon branches and refinements of glomerular tufts are observable (Figure 4A-E). Sensory neurons can be observed in vivo for extended time periods, e.g., longer than three weeks (Figure 4F-I). On the other hand, axons of immature neurons are still in the process of initial growth, do not possess tufted arborizations and have not yet connected to their final glomerular targets. The experimental protocol allows tracking the development of these neurons during maturation, e.g., axon elongation, bifurcations and establishment of tufted arborizations (Figure 4J-L). For more examples also see Hassenkl&#246;ver and Manzini12.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52143/52143fig1highres.jpg" width="500" /> 
Figure 1.&#160;Schematic overview of the experimental protocol. (A) Sensory neurons in the main olfactory epithelium (MOE) or vomeronasal organ (VNO) of the olfactory organ of anesthetized X. laevis larvae are labeled via electroporation using a glass pipette filled with fluorescent dextran solution. The axons of labeled neurons can be followed through the olfactory nerve (ON) and eventually reach the olfactory bulb (OB). (B) The tadpole is anesthetized and the labeled neurons are repeatedly investigated using a confocal or multiphoton microscope in specific time intervals. (C) The incremental development of axonal growth patterns of labeled cells can be followed over time spans of days to weeks.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52143/52143fig2highres.jpg" width="500" /> 
Figure 2.&#160;Electroporation of sensory neurons in the olfactory organ. (A) Electroporation with low resistance pipettes leads to labeling of multiple cells in the olfactory organ. In this representative example multiple olfactory receptor neurons (ORNs, filled arrowheads) and two columnar-shaped supporting cells (SCs, asterisks) were stained. Dashed lines demarcate the borders of the olfactory epithelium (OE). (B) Refining the electroporation parameters, e.g., increasing the pipette resistance, restricts the number of labeled cells. In this example, a single sensory neuron (filled arrowheads) and one adjacent supporting cell (asterisk) were stained after electroporation. Note the single axon leaving the olfactory epithelium (open arrowheads). (C) Successful single cell electroporation leads to exclusive labeling of an individual sensory neuron (filled arrowhead) and its connected axon (open arrowheads) in the olfactory epithelium. (D) The single labeled axon (open arrowheads) can be followed through the olfactory nerve (ON) into the olfactory bulb.
<img alt="Figure 3" fo:content-width="4in" src="/files/ftp_upload/52143/52143fig3highres.jpg" width="400" /> 
Figure 3.&#160;Visualizing olfactory axon projections in the excised olfactory bulb. (A) The coarse topology of axonal projections of sensory neurons in the olfactory bulb can be visualized using lower resistance electroporation pipettes. One olfactory bulb hemisphere and its associated olfactory nerve (ON) are depicted. Four dextrans coupled to different fluorophores were electroporated at four distant locations of the olfactory organ: lateral (green), intermediate (yellow), medial MOE (red) and VNO (orange). This allows visualizing the accessory olfactory bulb (AOB) and the three main projection fields of the main olfactory bulb (MOB). (B) Different axonal growth patterns of single olfactory sensory neurons superimposed on the structure of the olfactory bulb. Depicted are combined three dimensional reconstructions of multiple single cell stainings derived from different larval specimens. (C-E) Examples of single olfactory axons projecting into the olfactory bulb and forming tufted arborizations/synapses in spherical glomeruli (filled arrowheads). Note that in X. laevis olfactory axons bifurcate regularly (open arrowheads) before connecting to one, two or multiple glomeruli (filled arrowheads, also see Hassenkl&#246;ver and Manzini12).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/52143/52143fig4highres.jpg" width="500" /> 
Figure 4.&#160;In vivo time-lapse imaging of single olfactory neuron axons. (A-E) After successful single cell electroporation the labeled axon can be repeatedly visualized in the olfactory bulb (OB). This example shows an individual axon that was investigated for one week. The overall morphology does not change considerably and two major branching points can be identified (open arrowheads). Note that over the time course, one glomerular tuft undergoes continuous reduction (filled arrowhead), whereas the other two glomerular tufts remain stable (asterisks). (F-I) This representative example shows the viability of long-term observations as this specific axon was investigated for more than three weeks. No apparent change of its growth pattern can be detected. (J-L) An example of an immature sensory axon in the process of growth is depicted. It has not yet connected to glomeruli and characteristic glomerular tufts are missing. After 5 days the axonal branches are elongated, the axon bifurcated multiple times and fine arborizations are established.

Watch this Scientific Journal Video about The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo at JoVE.com

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

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

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

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

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Neuroscience

10.9K Views.  University of Göttingen. We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

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

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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 ...

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

Developmental Biology

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

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

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Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

Laser Nanosurgery of Cerebellar Axons In Vivo

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures