Funding provided by the National Eye Institute [5K08EY027850], National Institute of Child Health and Development [U54HD090255], Harvard-Vision Clinical Scientist Development Program [5K12EY016335], the Knights Templar Eye Foundation [Career Starter Grant], and the Children&#8217;s Hospital Ophthalmology Foundation [Faculty Discovery Award]. ECE is a Howard Hughes Medical Institute investigator.

All animal work described here was approved and performed in compliance with the Boston Children&#39;s Hospital Institutional Animal Care and Use Committee (IACUC) protocols.
1. Timed matings
<ol>
	<li>Place ISLMN:GFP (Islet Motor Neuron Green Fluorescent Protein; MGI: J:132726; Jax Tg(Isl-EGFP*)1Slp/J Stock No:&#160;017952) male and female mice together overnight. Weigh the females and record weights prior to mating. 
	NOTE: ISLMN:GFP specifically labels motor neurons with a farnesylated GFP that is not cytotoxic, localizes to the cell membrane of motor neurons and their axons, and allows the nerves to be visualized during development25. Other fluorescently labeled lines could also be used.</li>
	<li>Check for vaginal plugs in early morning. The date a plug is identified is designated as E0.5.</li>
	<li>Confirm pregnancy using weight gain and/or ultrasound at E10.5. Pregnant dams should have gained at least 1 g. Embryos can be seen on ultrasound at E10.5.</li>
</ol>
2. Preparation of&#160;reagents and vibratome for slice culture
<ol>
	<li>Prepare 500 mL of slicing buffer: add 5 mL of HEPES and 5 mL of penicillin/streptomycin to 500 mL of Hank&#8217;s Balanced Salt Solution (HBSS) without Ca2+ and Mg2+. &#160;Chill to 4 &#176;C. 
	NOTE: Extra slicing buffer should be stored at 4 &#176;C and can be used for future slice culture experiments.</li>
	<li>Prepare 50 mL of culture media: In a sterile hood, add 12.5 mL of HBSS, 12.5 mL of Fetal Bovine Serum, 250 &#181;L of glucose, 250 &#181;L of L-glutamine, 125 &#181;L of HEPES to 24.4 mL of Fluorobright DMEM. (Final concentrations: FlouroBright DMEM with 25% HBSS, 25% FBS, 0.5% glucose, 1 mM glutamine, and 2.5 mM HEPES.). &#160;Warm the culture media to 37 &#176;C in a sterile water bath. &#160; 
	NOTE: Culture media can be stored at 4 &#176;C for up to 3 weeks.
	<ol>
		<li>In a sterile hood, add 1.5 mL of culture media to each well of a 6-well plate. &#160;Add a cell culture insert (Table of Materials) to each well.&#160; Place in a sterile 37 &#176;C and 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>Prepare 4% low-melting temperature agarose: dissolve 2 g of low-melting temperature agarose in 50 mL of sterile PBS. Microwave in 30-60 s intervals until fully dissolved. Place in a 40 &#176;C water bath to keep liquid. 
	NOTE: Extra agarose can be stored at room temperature (RT) and melted for future slice culture experiments.</li>
	<li>Set up vibratome: Place a new blade on vibratome. Check settings: thickness 400-450 &#181;m. Pre-chill the vibratome stage. Place some ice in the outer chamber. Use a chamber and stage dedicated to live slices. Do not use the same vibratome chamber for fixed tissue as residual fixative could be damaging to slices.</li>
	<li>Prepare the microscope stage top incubator to 37 &#176;C and 5% CO2.</li>
	<li>Prepare for dissection: Clean surgical instruments and spray with 70% ethanol. Fill two Petri dishes with HBSS, place on ice. Open a 12 well tissue culture plate and place the lid on ice with the underside facing up. Open a 6 well tissue culture plate and place cell culture membrane inserts and 1.5 mL cell culture media in each well. Pre-warm the plate in a 37 &#176;C and 5% CO2 incubator.</li>
</ol>
3. Harvesting E10.5 embryos and preparing slices
NOTE: All steps from this point should be done as quickly as possible. Keep the embryos on ice at all times.
<ol>
	<li>Euthanize the pregnant dam (E10.5) in a CO2 chamber. Perform cervical dislocation.</li>
	<li>Spray the abdomen with 70% ethanol. Cut open the abdomen with scissors, remove the uterus and place it in a Petri dish with ice cold HBSS to quickly wash away blood. Move the washed uterus to a second Petri filled with dish ice cold HBSS.</li>
	<li>Under a dissecting scope, remove the embryos from the uterine horn and individual amniotic sacs. Place the embryos on the underside of the lid of a 12 well plate. Keep on ice.</li>
	<li>Under a dissecting scope, use filter paper to remove any liquid surrounding each embryo. 
	NOTE: Embryos will stick to the filter paper if touched.</li>
	<li>Embed embryos in agarose. Pour melted agarose over each embryo to cover it. Keep on ice. As soon as the agarose has hardened, flip each embryo and pour additional agarose on the other side. Keep on ice.</li>
	<li>Using a fluorescent dissecting scope, trim the agarose around each embryo so it will be oriented properly when glued to the vibratome stage. The oculomotor nucleus and early axon outgrowth are fluorescent. Align the embryo so that the nucleus, outgrowing axons, and eye form a line, and trim the agarose with a razor blade cut parallel to this line (dorsal to the embryo, see Figure 1A). 
	NOTE: This will be the side glued to the vibratome stage (the embryo will be positioned on its back, head closest to the vibratome blade). A line between the oculomotor nucleus and eye should be parallel to the vibratome blade.</li>
	<li>Fill the vibratome chamber with ice-cold slice buffer. Superglue the embryo to the vibratome stage so that the blade will be parallel with the oculomotor nucleus and eyes. Once the superglue is dry, submerge the vibratome stage so the embryo is oriented facing away from blade.</li>
	<li>Slice 400-450 &#181;m slices.&#160; Collect each slice with a sterile transfer pipet. Place into cold slicing buffer.</li>
	<li>Under the dissecting scope, choose the slice containing the oculomotor nuclei and eyes. Using a sterile transfer pipet, place it on the cell culture insert in the 6 well plate. Return the plate to the 37 &#176;C incubator. Alternatively, have one person slicing and another placing the slices. Minimize the time between slicing and placing into incubator. 
	NOTE: Slices should be oriented in a way that the maximum fluorescence emitted from the nuclei and axons is closest to the imaging microscope objective. On an inverted microscope, the slices should be placed on the membrane with the nuclei and axons closest to the objective underneath the plate.</li>
	<li>Remove the residual agarose from the vibratome stage, superglue the next embryo to the stage and repeat steps 3.7-3.9 until all embryos have been sliced and plated.</li>
	<li>Add inhibitor or recombinant molecule of choice to media in each well to create a dose-response curve.&#160; Dilute in appropriate solvent. 
	NOTE: If using DMSO, use only tissue culture grade DMSO. Alternatively, protein-eluting beads can be placed in specific locations on the slice.</li>
	<li>Place on the microscope in the 37 &#176;C and 5% CO2 chamber.&#160; Set the microscope to take phase contrast and fluorescent photographs of each slice every 30 min (or more often if desired). Slices can be maintained for 48-72 h.</li>
</ol>

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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=Neuropilin-2+is+required+in+vivo+for+selective+axon+guidance+responses+to+secreted+semaphorins.">Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.</a> Neuron. 25 (1), 29-41 (2000).</li><li>Chen, H., 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=Neuropilin-2+regulates+the+development+of+selective+cranial+and+sensory+nerves+and+hippocampal+mossy+fiber+projections.">Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.</a> Neuron. 25 (1), 43-56 (2000).</li><li>Lerner, O., 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=Stromal+cell-derived+factor-1+and+hepatocyte+growth+factor+guide+axon+projections+to+the+extraocular+muscles.">Stromal cell-derived factor-1 and hepatocyte growth factor guide axon projections to the extraocular muscles.</a> Developmental Neurobiology. 70 (8), 549-564 (2010).</li><li>Cheng, L., 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=Human+CFEOM1+mutations+attenuate+KIF21A+autoinhibition+and+cause+oculomotor+axon+stalling.">Human CFEOM1 mutations attenuate KIF21A autoinhibition and cause oculomotor axon stalling.</a> Neuron. 82 (2), 334-349 (2014).</li><li>Tischfield, M. A., 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=Human+TUBB3+mutations+perturb+microtubule+dynamics,+kinesin+interactions,+and+axon+guidance.">Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.</a> Cell. 140 (1), 74-87 (2010).</li><li>Kim, M., 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=Motor+neuron+cell+bodies+are+actively+positioned+by+Slit/Robo+repulsion+and+Netrin/DCC+attraction.">Motor neuron cell bodies are actively positioned by Slit/Robo repulsion and Netrin/DCC attraction.</a> Developmental Biology. 399 (1), 68-79 (2015).</li><li>Montague, K., Guthrie, S., Poparic, I. <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+and+In+Vitro+Knockdown+Approaches+in+the+Avian+Embryo+as+a+Means+to+Study+Semaphorin+Signaling.">In Vivo and In Vitro Knockdown Approaches in the Avian Embryo as a Means to Study Semaphorin Signaling.</a> Methods in molecular biology. 1493, 403-416 (2017).</li><li>Clark, C., Austen, O., Poparic, I., Guthrie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha2-Chimaerin+regulates+a+key+axon+guidance+transition+during+development+of+the+oculomotor+projection.">alpha2-Chimaerin regulates a key axon guidance transition during development of the oculomotor projection.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33 (42), 16540-16551 (2013).</li><li>Ferrario, J. 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The authors have nothing to disclose.

This ex vivo slice culture protocol provides significant advantages over traditional axon guidance assays23. The size of each cranial motor nucleus is not a limiting factor, and no difficult dissection is necessary. The endogenous microenvironment through which the axons travel is maintained, allowing modification of one signaling pathway while maintaining other signaling pathways. Additionally, effects can be assessed at different points along the axon trajectory. Since axon guidance requires mul...

The ocular motor system provides an elegant system for investigating axon guidance mechanisms. It is relatively uncomplicated, consisting of three cranial nerves innervating six extraocular muscles (EOMs) which move the eye, and the levator palpebrae superioris (LPS) which lifts the eyelid. The oculomotor nerve innervates the LPS and four EOMs - the inferior oblique and the medial, inferior, and superior rectus muscles. The other two nerves, the trochlear and abducens, each only innervate one muscle, the superior oblique and lateral rectus muscle, respectively. Eye movements provide an easy readout, showing if innervation was appropriate, missing, or aberrant. Additionally, there are human eye movement disorders that result from deficits in neuronal development or axon guidance, collectively termed the congenital cranial disinnervation disorders (CCDDs)1.
Despite these advantages, the ocular motor system is rarely used in axon guidance studies2,3,4,5,6,7,8,9,10, due to technical drawbacks. In vitro axon guidance assays have many disadvantages11. Co-culture assays, in which neuronal explants are cultured together with explants of target tissue12 or transfected cells13, depend on both symmetry of the explant and precise positioning between the explant and target tissue. Stripe assays14,15, in which two cues are laid down in alternating stripes and axons are assessed for preferential growth on one stripe, only indicate that one substrate is preferable to the other, not that either is attractive or repulsive, or physiologically relevant. Microfluidics chambers can form precise chemical gradients, but subject growing axons to shear stress16,17,18, which can affect their growth. Moreover, in each of these approaches, collecting explants or dissociated cells requires that outgrowing axons be axotomized and thus these assays actually examine axon regeneration, rather than initial axon outgrowth. Finally, these in vitro approaches remove the microenvironment that influences axons and their responses to cues along different points of their course, and traditionally only test one cue in isolation. Compounding these disadvantages, the small size of each nucleus in the ocular motor system makes dissection technically challenging for either explants or dissociated cultures. Additionally, primary cultures of ocular motor neurons are usually heterogeneous, have significant cell death, and are density dependent, requiring pooling of cells from multiple embryos (Ryosuki Fujiki, personal communication). In vivo methods, however, including knockout mouse models, are inappropriate to use for screening, given the time and expense required.
Methods developed to culture whole embryos19 allow labeling of migrating cells20 or blockade of specific molecules21, but whole embryo cultures require incubation in roller bottles which precludes real-time imaging of labeled structures. Surgical techniques that allow manipulation of the embryo and then subsequent further development either in the uterus or in the abdomen of the mother (maintaining the placental connection)22 are also possible, but these also do not allow time-lapse imaging.
To overcome the obstacles of in vitro assays and allow rapid screening of signaling pathways, an ex vivo embryonic slice culture technique was developed23, adapted from a previously published protocol for peripheral nerve outgrowth24. Using this protocol, the developing oculomotor nerve can be imaged over time in the presence of many of the surrounding structures along its trajectory, including EOM targets. By adding small molecule inhibitors, growth factors, or guidance cues to the culture media, we can assess guidance perturbations at multiple points along the axon trajectory, allowing more rapid assessment of potential growth and guidance factors.

<table border="0" cellpadding="0" cellspacing="0" style="width:1196px;" width="1196">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">24-Well Tissue Culture Plate</td>
			<td style="width:163px;">Genesee Scientific</td>
			<td style="width:344px;">25-107</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6-Well Tissue Culture Plate</td>
			<td>Genesee Scientific</td>
			<td>25-105</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable Pasteur Pipet (Flint Glass)</td>
			<td>VWR</td>
			<td>14672-200</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11412-11</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorobrite DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1896701</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose (200 g/L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2494001</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#39;s Balanced Salt Solution (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-095</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550H</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES Buffer Solution (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630106</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Glutamine (250 nM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Loctite Superglue</td>
			<td>Loctite</td>
			<td></td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Low Melting Point Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>16520050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um)</td>
			<td>Millipore Sigma</td>
			<td>PICM03050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Moria Mini Perforated Spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-19</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122&#160;</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri Dish (100 x 15mm)</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphate Buffered Saline (1X, pH 7.4)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical Scissors - Blunt</td>
			<td>Fine Science Tools</td>
			<td>14000-12</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ti Eclipse Perfect Focus with TIRF</td>
			<td>Nikon</td>
			<td></td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome (VT 1200S)</td>
			<td>Leica</td>
			<td>1491200S001</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Blades (Double Edge, Stainless Steel)</td>
			<td>Ted Pella, Inc.</td>
			<td>121-6</td>
			<td style="width:331px;"></td>
		</tr>
	</tbody>
</table>

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.

Normal Results: Figure 1 provides a schematic of the experiment. Starting as early as E9.5 in mouse, the first axons begin to emerge from the oculomotor nucleus26. By E10.5, a fasciculated oculomotor nerve, which contains the early pioneer neurons, can be seen in the mesenchyme. There is significant variability between embryos at E10.5 (even within the same litter) in how far the nerve has progressed towards the orbit, likely due to developmental differences of a few ...

Watch this Scientific Journal Video about Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth at JoVE.com

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

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 ...

ex-vivo-oculomotor-slice-culture-from-embryonic-gfp-expressing-mice

Research

JoVE Journal

Neuroscience

8.3K Views.  Boston Children's Hospital. 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.

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

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...