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. 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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><tbody><tr><td>24-Well Tissue Culture Plate</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>6-Well Tissue Culture Plate</td><td>Genesee Scientific</td><td>25-105</td><td></td></tr><tr><td>Disposable Pasteur Pipet (Flint Glass)</td><td>VWR</td><td>14672-200</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11412-11</td><td></td></tr><tr><td>Fluorobrite DMEM</td><td>Thermo Fisher Scientific</td><td>A1896701</td><td></td></tr><tr><td>Glucose (200 g/L)</td><td>Thermo Fisher Scientific</td><td>A2494001</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (1X)</td><td>Thermo Fisher Scientific</td><td>14175-095</td><td></td></tr><tr><td>Heat Inactivated Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11550H</td><td></td></tr><tr><td>HEPES Buffer Solution (1M)</td><td>Thermo Fisher Scientific</td><td>15630106</td><td></td></tr><tr><td>L-Glutamine (250 nM)</td><td>Thermo Fisher Scientific</td><td>25030081</td><td></td></tr><tr><td>Loctite Superglue</td><td>Loctite</td><td></td><td></td></tr><tr><td>Low Melting Point Agarose</td><td>Thermo Fisher Scientific</td><td>16520050</td><td></td></tr><tr><td>Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um)</td><td>Millipore Sigma</td><td>PICM03050</td><td></td></tr><tr><td>Moria Mini Perforated Spoon</td><td>Fine Science Tools</td><td>10370-19</td><td></td></tr><tr><td>Penicillin/Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140122&amp;nbsp;</td><td></td></tr><tr><td>Petri Dish (100 x 15mm)</td><td>Genesee Scientific</td><td>32-107G</td><td></td></tr><tr><td>Phosphate Buffered Saline (1X, pH 7.4)</td><td>Thermo Fisher Scientific</td><td>10010049</td><td></td></tr><tr><td>Razor Blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Surgical Scissors - Blunt</td><td>Fine Science Tools</td><td>14000-12</td><td></td></tr><tr><td>Ti Eclipse Perfect Focus with TIRF</td><td>Nikon</td><td></td><td></td></tr><tr><td>Vibratome (VT 1200S)</td><td>Leica</td><td>1491200S001</td><td></td></tr><tr><td>Vibratome Blades (Double Edge, Stainless Steel)</td><td>Ted Pella, Inc.</td><td>121-6</td><td></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 ...

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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

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Neuroscience

8.4K Views.  Boston Children's Hospital. Dit protocol stelt ons in staat om axon begeleidingstrajecten actief in de oculomotorische zenuw te identificeren en om hun rollen te beoordelen op verschillende punten langs het zenuwtraject in real time. Deze techniek behoudt de lokale omgevingen waardoor axonen reizen en hun uiteindelijke doelen. De groeiende axonen worden niet gesneden, dus initiële axon uitgroei, in plaats van regeneratie, kan worden beoordeeld.Deze methode geeft inzicht in axon begeleiding in het oculaire motors...

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 ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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 ...

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 ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Time-Lapse Imaging of Cortical Neuron Radial Migration in Transduced Mouse Embryonic Brain Slices

Source:<a style='text-decoration:none;' href='https://dx.doi.org/10.3791/55886-v'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Wiegreffe, C.,&#160;et. al.,&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using&#160;In Utero<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Electroporation.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2017)</a> &#160;This video demonstrates time-lapse confocal microscopy to observe cortical neuron radial migration from the subventricular zone to the cortical plate via radial glial scaffolds in transduced mouse embryonic brain slices.

Source:&#160;Wiegreffe, C.,&#160;et. al.,&#160;&#160;Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse ...

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

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