We thank Dan McWhorter for narrating the protocol in the video. This work is supported by NIH Grant
5R01NS062182, a grant from American Federation for Aging Research, and institutional funds awarded to HTG.

I.	Procedures
The following techniques should be performed under sterile conditions, in a laminar flow hood, using sterilized tools.
Preparation of glass bottom dishes for organotypic slices
<ol>
<li>Dishes must be prepared in sterile environment and using sterilized tools.</li>
<li>A 150&mu;L drop of slice medium (see recipes) is placed in the center of the glass-bottom part of the dish with care to avoid air bubbles in the medium.</li>
<li>With a disposable syringe equipped with a 23 Gauge needle, multiple spots of glue (Rubber Cement - Elmer's, cat# E904) are placed on the square fringes adjacent to the circular coverslip that occupies the center of the culture dish, while leaving one side unglued for fluid exchange (Figure 1). The listed type of glue is not toxic to the slices or the cells as applied in this protocol. Care must be given to avoid placing any glue on the glass cover slip as this will impinge upon and obstruct imaging of the slices later. A nucleopore membrane (diameter 25mm, pore size 8.0&mu;m - Whatman, cat# 110614) is placed on top of the glass coverslip, using the glue spots to secure it in place. This should be done using a micro forceps, while ensuring that air bubbles are not trapped between the glass coverslip and the membrane.</li>
<li>Add 1 mL slice medium on top of membrane. Dishes are placed in an incubator for 30 minutes and then on ice, until ready to use.</li>
</ol>
Extraction of early postnatal brains
The best results are obtained when slices are prepared from young postnatal mice (P1-P10).
<ol>
<li>Pups are terminally anesthetized (overdosed) by isofluorane or other approved methods. The head can be sprayed with 70% ethanol to increase sterility, followed by rapid decapitation using sharp scissors.</li>
<li>The head is stabilized by clamping the mandible with micro forceps. The skin is excised longitudinally from the neck to the snout. The skull is cut longitudinally and anteriorly starting at the cisterna magna, by making 1 medial and 2 lateral cuts (one on each side - Figure 2A). Care should be taken to minimize contact with underlying cortical tissue as the cranial flaps are removed away from the brain.</li>
<li>To improve stability of the tissue during vibratome sectioning, the lateral-most aspects of the brain are removed by making two sagittal cuts. The caudal aspect of the brain is also removed by making a cut at the rostral base of the cerebellum (Figure 2B).</li>
<li>The two hemispheres are separated by making a smooth cut along the midline fissure, and the two hemispheres are carefully scooped out of the skull and placed, medial surface down, in an embedding mold (Figs. 2C-D).</li>
</ol>
Sectioning of the host brain
<ol>
<li>The two hemispheres in the embedding mold are immediately covered with melted 3% low melting point DNA-grade agarose gel (Fisher, cat# BP1360-100) dissolved in tissue preparation buffer that is maintained at 37&deg;C (see recipes). After 2 minutes of stabilization on a flat horizontal surface to ensure even hardening of the agarose, the molds are positioned on ice to complete the setting.</li>
<li>Once the gel containing the hemispheres is set, it is removed from the mold and trimmed, leaving 2-3mm of gel around the brain tissue.</li>
<li>The gel-embedded tissue is then mounted on the specimen disc of the vibratome, with the medial surface up, and secured with cyanoacrylate adhesive (Krazy glue or equivalent). Care must be taken to apply only minimal amounts of glue, as too much will have toxic effects on slices and migrating cells. Too much glue on the sides of the block will also impede the cutting, causing potential damage in the tissue.</li>
<li>The disc is installed in the vibratome specimen tray filled with ice-cold tissue preparation medium.</li>
<li>The tissue is sectioned at 150&mu;m thickness, with the vibratome speed set on a slow-to-medium range (will depend on the vibratome used, and thus must be determined independently for optimal results). In our hands, the vibration frequency is optimal when set at maximum. The first few slices may be discarded.</li>
<li>As soon as the RMS-containing slices are released from the blade (the RMS is visible by the naked eye as a grey U-shaped structure extending from the SEZ to the OB), they are carefully teased out of the gel mold, and scooped using a small flathead spatula. The slices are placed on the nucleopore membrane of the glass-bottom dishes containing slice medium (Figure 3A-B). A typical early postnatal mouse brain will yield approximately 2-3 RMS sections per hemisphere when cut at 150 &mu;m. It is critical that handling the slices is kept to a minimum as they are very fragile. The dishes with the slices are then transferred to an incubator.</li>
</ol>
Donor brain sectioning and RMS transplantation
<ol>
<li>Donor brains (brains expressing fluorescent reporters in migrating neuroblasts) are sectioned at 250&mu;m thickness and slices are collected in ice-cold tissue preparation buffer.</li>
<li>Slices are immediately placed under a dissecting microscope with epifluorescence capability, and the RMS is gently microdissected, using micro forceps. One forceps is used to stabilize the slice while the other is used to make small cuts around the RMS until it is released from the slice. The excised RMS is then cut into small explants (approximately 200-500 &mu;m in diameter) prior to transplantation.</li>
<li>Glass-bottom dishes containing host slices positioned on the nucleopore filters are removed from the incubator and placed under the dissecting microscope. Using visible light, the RMS is clearly identified and a small incision is made in the initial segment of the RMS.</li>
<li>Using a pipettor equipped with a 20 &mu;L tip, a single donor RMS explant is transferred to the incised site in the host RMS. The explant is gently pushed into the incision to establish contact between the 2 tissues. To ensure this contact is stable, the explants are pushed slightly in between the slice and the nucleopore membrane.</li>
<li>Once all host slices with the RMS are transplanted, the dishes are returned to the incubator for at least 1 hour to allow the sections to settle on the membrane. Neuroblasts should begin to migrate from the explant into the host RMS after approximately 1-2 hours.</li>
</ol>
Time-lapse imaging of neuronal migration
<ol>
<li>The glass-bottom dishes are transferred from the incubator to the incubated chamber on the microscope (Figure 3C). Fluorescent neuroblasts can be imaged at intervals ranging from 0 to 10 minutes depending on the type of analysis desired. For example we image cytoskeletal dynamics during the migratory cycle of individual neuroblasts by setting our intervals at less than or equal to 2.5 minutes. However, population dynamics such as orientation and speed of migration are better captured at intervals ranging from 5 to 10 minutes. The choice of objectives is highly variable depending on the brand of microscope. For a Nikon C1 confocal microscope, we find that the 20x dry lens (Nikon Pan Fluor, N.A. 0.75, W.D. 0.35mm) is most suitable for our analyses. For best results on this confocal system, the pinhole is opened to a medium size (60&mu;m diameter). For most appropriate migratory behavior, imaging is confined to cells deep within the thickness of the RMS, at least 20 &mu;m away from either of the cut surfaces of the slice. Laser power should be optimized so that it is at a minimum, but that the details of individual neuroblasts remain visible.</li>
<li>Once the imaging is completed slices can be fixed with ice cold, and freshly prepared 4% paraformaldehyde, immunostained, and mounted on slides for further imaging. Since we do not coat our membranes with any adherent substrates such as laminin, the slices usually float away from the membrane once they are immersed in the staining buffers. Sections are imaged while maintaining their 150 &mu;m thickness. It is also possible to cryopreserve and freeze the slices and use a cryostat to obtain thinner sections of the original slices. However, this will result in increase the incidence of artifacts in staining as well as alter the integrity of the tissue.</li>
</ol>
II.	 Materials/equipment
Preparation of glass bottom dishes for organotypic slices
<ul>
<li>Small syringes (1 mL)</li>
<li>23-gauge needles</li>
<li>Nucleopore Track-Etch Membrane - diameter 25mm, pore size 8.0&mu;m - Whatman, cat# 110614</li>
<li>Glass Bottom Culture Dishes - 35mm petri dish, 14mm Microwell, No. 1.5 coverglass - MatTech, cat# P35G-1.5-14-C</li>
<li>Rubber Cement - Elmer's, cat# E904</li>
<li>Basal Medium Eagle - Gibco, cat# 21010</li>
<li>1M Hepes (pH7.4)</li>
<li>1M D-glucose</li>
<li>100mM CaCl2</li>
<li>100mM MgSO4</li>
<li>1M NaHCO3</li>
<li>dH2O</li>
<li>200mM L-glutamine</li>
<li>Penicillin-streptomycin</li>
</ul>
Brain extraction and embedding
<ul>
<li>Anesthetic (isofluorane, etc.)</li>
<li>Microwave oven</li>
<li>Low Melting Agarose - Fisher, cat# BP1360-100</li>
<li>Krazy glue - cat#KG585</li>
<li>Peel-A-Way Disposable Embedding Molds (R-40) - 22mmx40mm rectangular, 20mm deep - Polysciences, cat# 18646C</li>
</ul>
Brain sectioning and RMS transplantation
<ul>
<li>Vibratome - Leica VT1000S and all accessory components for slice preparation</li>
<li>10X Hank's Balanced Salt Solution - Gibco, cat# 14185</li>
<li>Microdissecting forceps #5 - Roboz, cat# RS-4976</li>
<li>Microspatula - Fisher, cat# 21-401-15</li>
<li>Stereomicroscope</li>
</ul>
Time-lapse imaging of organotypic slices
<ul>
<li>Humidified Incubator, 5% CO2</li>
<li>Inverted Microscope equipped with incubator chamber and long distance working objectives (NA of 0.6 or higher)</li>
</ul>
III.	Recipes
Buffer solution for tissue dissection and slice preparation (tissue preparation buffer)
<table border="1">
<tbody>
 <tr>
 <td>Stock solution</td>
 <td>Volume</td>
 <td>Final Concetration</td>
 </tr>
 <tr>
 <td>10X HBSS</td>
 <td>50 mL</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>1M Hepes (pH 7.4)</td>
 <td>1.25 mL</td>
 <td>2.5mM</td>
 </tr>
 <tr>
 <td>1M D-Glucose</td>
 <td>15 mL</td>
 <td>30mM</td>
 </tr>
 <tr>
 <td>1M CaCl2</td>
 <td>0.5 mL</td>
 <td>1mM</td>
 </tr>
 <tr>
 <td>1M MgSO4</td>
 <td>0.5 mL</td>
 <td>1mM</td>
 </tr>
 <tr>
 <td>1M NaHCO3</td>
 <td>2 mL</td>
 <td>4mM</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>430.75 mL</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Filter sterilize with a 0.2 &mu;m filter and store at 4&deg;C.

Culture medium for organotypic slices, tissue transplantation and imaging (slice medium)
<table border="1">
<tbody>
 <tr>
 <td>Stock solution</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Basal Medium Eagle</td>
 <td>35 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue preparation buffer</td>
 <td>12.9 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M D-Glucose</td>
 <td>1.35 mL</td>
 <td>20mM</td>
 </tr>
 <tr>
 <td>200mM L-glutamine</td>
 <td>0.25 mL</td>
 <td>1mM</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>0.5 mL</td>
 <td>100units/ mL penicillin and 0.1mg/ mL streptomycin</td>
 </tr>
 </tbody>
</table>

Filter sterilize with a 0.2 &mu;m filter and store at 4&deg;C.

Preparation of Low-melting point agarose gel
Low-melting-point agarose is diluted in tissue preparation buffer at 0.3g/ mL in a 50 mL conical tube (see recipes). The tube is microwaved in increments of 5-10 seconds at high-power. Number of increments is dependent on total volume; for 10 mL, three increments (10-8-5 seconds, each) should suffice. The tube cap is carefully unscrewed between heating increments to release air pressure and avoid explosion of the tube. Caution must be taken as the tube content will be very hot. Once the agarose is completely dissolved, the tube is maintained in a 37&deg;C water bath for at least 5 minutes to allow the temperature to stabilize prior to use. Prolonged exposure to room temperature will harden the gel. Although this must be avoided as much as possible, hardened gel can be reheated and re-melted for immediate use within 24 hours of initial preparation.
Immunohistochemistry on organotypic slices
Following imaging on the confocal microscope, slices may be fixed overnight at 4&deg;C with 4% formaldehyde in PBS. Sections are then blocked overnight at 4&deg;C, in 10% goat serum with 1% Triton X (Sigma, Cat. # S26-36-23) in PBS followed by overnight incubation with primary antibodies at 4&deg;C. Fluorescently-tagged goat secondary antibodies are used for visualization (all diluted 1:1000, 1 hour incubation at room temperature). Labeled slices are thoroughly washed 5-6 times with ice cold PBS prior to mounting on glass slides and coverslipping.
IV. Representative Results
Our organotypic slice culture protocol has been thoroughly tested and optimized over that last few years for consistency in migration pattern and orientation. Analysis of cells emigrating from explants obtained from mice in which the expression of the red fluorescent protein, Td-tomato, is induced under the Nestin promoter (Nestin-Td tomato), reveals highly oriented and rapid migration of tdTomato+ neuroblasts into the host RMS (Figure 4A). High-magnification time-lapse analysis illustrates excellent resolution of the entire length of a migrating neuroblast during a 20-minute imaging session (Figure 4B).
Slices with donor Td-tomato+ cells were fixed and immunostained for different cellular components within the RMS. GFAP+ astrocytes and CD31+ blood vessels were revealed using fluorescent immunohistochemistry (Figure 5A). High-magnification analysis of slices stained for the cytoskeletal proteins actin and tubulin reveal non-uniform expression of these components by a cell in the midst of migration (Figure 5B).
Antibodies used in these examples: rabbit anti-RFP (Abcam, 1:250), rabbit anti-GFAP (Dako, 1:1000), rat anti-CD31(BD Pharmigen, 1:100), mouse anti-actin (Santa Cruz, 1:500), rabbit anti-tubulin (Sigma, 1:1000), goat anti-mouse Cy3 (Chemicon, 1:1000), goat anti-rabbit AlexaFluor 647 (Invitrogen, 1:1000), goat anti-rat AlexaFluor 488 (Invitrogen, 1:1000), goat anti-rabbit AlexaFluor 488 (Invitrogen, 1:1000).
<img src="/files/ftp_upload/2486/2486fig1.jpg" alt="Figure 1" /> 
Figure 1. Preparation of glass-bottom dishes for organotypic slices. Multiple spots of glue are placed around the circular glass-bottom component of the dish (red), leaving one side open for exchange of medium from underneath the filter. A 150&mu;L drop of slice medium is placed in the center of the glass coverslip. A nucleopore membrane (blue) is then applied, shiny side down, while ensuring that no air bubbles are trapped between the glass coverslip and the membrane. One milliliter of slice medium (grey) is spread on top of membrane, and dishes are incubated at 37&deg; C prior to use.
<img src="/files/ftp_upload/2486/2486fig2.jpg" alt="Figure 2" /> 
Figure 2. Brain extraction and preparation for sectioning. (A) The skull is exposed by incising the scalp from the neck to the snout (dotted line along the midline). The skull is then cut longitudinally and anteriorly starting at the cisterna magna, by making 1 medial and 2 lateral cuts (one on each side; 2A). (B) The lateral-most aspects of the cortex and the caudal aspect of the CNS are resected to improve stability of the tissue during vibratome sectioning. (C-D) The two hemispheres are then separated and placed medial face down in an embedding mold prior to application of 3% agarose gel dissolved in tissue preparation buffer.
<img src="/files/ftp_upload/2486/2486fig3.jpg" alt="Figure 3" /> 
Figure 3. Brain sectioning and cross-transplantation. (A) The host tissue is sectioned at 150&mu;m thickness, and the RMS-containing sections are carefully positioned flat on the nucleopore membrane of cold glass-bottom dishes. (B) Donor brains (brains expressing fluorescent reporters in the RMS) are sectioned at 250&mu;m thickness, and the slices are collected in ice-cold tissue preparation buffer. The donor RMS is microdissected and cut into small explants. Using a pipettor equipped with a 20 &mu;L tip, individual RMS explants are transferred to an incised site in the host RMS. (C) After 1-2 hours of incubation, dishes are transferred to an incubated stage on a confocal microscope and migration is captured using time-lapse imaging. The photomicrograph is a representative low-magnification image of a typical slice (grey) set up 1 hour after transplantation (red explant from tdTomato+ RMS of a donor mouse; red dotted lines outline the RMS in the host slice).
<img src="/files/ftp_upload/2486/2486fig4.jpg" alt="Figure 4" /> 
Figure 4. Migration of neuroblasts from explants into the host RMS. (A) Nestin-tdTomato+ neuroblasts (red) migrate from the explants into the host RMS (dotted green line) 1 hour after transplantation. tdTomato+ cells invading the RMS of the host organotypic slices move in a highly oriented and rapid manner away from the SEZ and toward the OB. (B) The migratory cycle can be observed in high-power time-lapse images of a neuroblast over approximately a 20 minute period. Scale bar = 10 &mu;m.
<img src="/files/ftp_upload/2486/2486fig5.jpg" alt="Figure 5" /> 
Figure 5. Immunohistochemical assessment of organotypic slices. Explanted neuroblasts (red) were fixed in the midst of migration 12 hours post transplantation. (A) Fluorescent immunohistochemical staining of the slice reveals a dense pool of GFAP+ astrocytes (blue) and scattered CD31+ blood vessels (green) within the RMS of the host slice. (B) The cytoskeleton of an isolated tdTomato+ migrating cell (red) in a host RMS is revealed by co-immunostaining using antibodies against actin (blue) and tubulin (green).

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Experiments on animals were performed in accordance with the guidelines and regulations set forth by North Carolina State University and its College of Veterinary Medicine.
No conflicts of interest declared.

Neuronal migration in the RMS is an essential component of postnatal neurogenesis in the olfactory bulbs 1. Migration through the RMS occurs in a plane tangential to the surface of the brain. Tangentially migrating neuroblasts are distinct from radially migrating cells based on the location of their progenitor source, as well as the divergent fate of their final neuronal products 1, 2, 3. The relatively pure population of tangentially migrating cells in the postnatal RMS makes this anatomically de...

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.

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

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 ...

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Neuroscience

11.9K Views.  North Carolina State University. 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.

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

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 ...

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 ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

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 ...

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 ...

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

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

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

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

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 ...

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

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Chapters in this video

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

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

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

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

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

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

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

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

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

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