Name: the olfactory system as a model to study axonal growth patterns and morphology in vivo
Uploaded: 2014-10-30T15:00:00
Duration: 8 min 29 s
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>

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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 border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Name of Material/ Equipment</td>
			<td style="width:100px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">SZX16</td>
			<td style="width:100px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">stereomicroscope with fluorescent illumination</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Axon Axoporator 800A</td>
			<td style="width:100px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">single cell electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">ELP-01D</td>
			<td style="width:100px;">npi electronic</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">MMJ</td>
			<td style="width:100px;">M&#228;rzh&#228;user Wetzlar</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">manual micromanipulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">P-1000</td>
			<td style="width:100px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Horizontal micropipette puller</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:203px;">G150F-4</td>
			<td style="width:100px;">Warner Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">glass capillaries for electroporation pipette fabrication; internal filament makes backfilling easier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 488-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22910</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 546-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22911</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 568-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22912</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 594-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22913</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">TMR-dextran 3kD (micro-Ruby)</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D7162</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">microloader pipette tips</td>
			<td style="width:100px;">eppendorf</td>
			<td style="width:119px;">930001007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">E10521</td>
			<td style="width:167px;">anesthetic; use gloves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">A1-MP</td>
			<td style="width:100px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiphoton microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">LSM 780</td>
			<td style="width:100px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">confocal microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Imaris</td>
			<td style="width:100px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">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

A Caenorhabditis elegans Model System for Amylopathy Study

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A Caenorhabditis elegans Model System for Amylopathy Study

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Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

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Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

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Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

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A Free-breathing fMRI Method to Study Human Olfactory Function

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We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

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Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
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Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

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Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

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This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

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Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

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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.
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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 ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...