Name: Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro (Video) | JoVE Journal
Uploaded: 2012-01-31T12:00:00.000+00:00
Duration: 7 min 33 s
Description: Learn organotypic slice culture of E12.5 murine embryonic midbrain for studying dopaminergic neuron development in vitro. This method enables time-lapse imaging of neuronal migration and differentiation.

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5 minutes

We thank Martine Emond and Isabel Brachmann for their help in establishing the organotypic slice culture system and Wolfgang H&uuml;bner and Liviu Gabriel Bodea for critical reading of the manuscript. We would like to thank Frank Costantini for the R26 reporter mice and Cliff Tabin for the ShhCreER mice. This study was funded by a Research Award from the Ministry of Science and Research of North-Rhine Westphalia (Programm zur F&ouml;rderung der R&uuml;ckkehr des wissenschaftlichen Spitzennachwuchses aus dem Ausland).

Parts of this protocol are modified from Daza et al., 2007 7.
1. Preparations
<ol>
	<li>Can be prepared one day in advance
	<ol>
		<li>Prepare 1X Krebs buffer (1.5 L): 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4:H2O, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose, 25 mM NaHCO3; adjust pH to 7.4. Filter sterilize (0.22 μm pore size) and store at 4°C.</li>
		<li>Prepare the culture medium (20 mL): 5 mL HBSS, 9 mL DMEM high glucose, 850 μL of 30 % glucose, 5 mL horse serum (25 %). Add 200 μL 100X Penicllin/Streptomycin. Store at 4°C.</li>
	</ol>
	</li>
	<li>Prepare before starting the dissection
	<ol>
		<li>Prepare 100 mL of 4 % low melting agarose (LMP agarose) in 1X Krebs buffer: microwave the solution until the agarose is completely dissolved and then place the agarose in a 45 °C water bath.</li>
		<li>Fill up the vibratome buffer tray with ice-cold 1X Krebs buffer and start the cooling element (keep at 4°C). Fix a razor blade in the blade carrier and set-up the vibratome area with a scalpel, a fine paintbrush and a mini perforated spoon to pick up the slices. Prepare sterile Petri dishes (35 x 10 mm) with 1x Krebs buffer for collecting the slices. Keep on ice.</li>
		<li>Set-up the dissection area with sterile Petri dishes (100 x 15 mm) for dissection and smaller sterile Petri dishes (35 x 10 mm) for embedding, small scissors, two fine forceps (Dumont 5), a mini perforated spoon, a glass Pasteur pipette fire polished with a round closed tip and 1 L of 1X Krebs buffer on ice. Wipe all the dissection tools with 70 % ethanol.</li>
		<li>Add culture medium to the wells of a six-well plate (1.5 mL/well) and place it in a 37 °C incubator.</li>
		<li>Prepare a six-well plate with 1.5 mL/well sterile 1X Krebs buffer and 15 μL Penicilin/Streptomycin (100X)/well. Under sterile conditions, place Millicell Cell Culture inserts into the wells. Place the six-well plate next to the vibratome so that the brain slices can be transferred onto the filter membranes immediately after sectioning.</li>
	</ol>
	</li>
</ol>
2. Dissection and embedding of embryonic brains
<ol>
	<li>Anesthetize a pregnant female mouse using isoflurane and sacrifice the mouse by cervical dislocation (embryos should be at stage E12.5). Dissect out the uterus of the mouse by pulling up the uterus with forceps. Use other forceps to separate the mesometrium away from the uterus. Place the uterus in ice-cold 1X Krebs buffer. Use a fine forceps to separate the muscular wall of the uterus, Reichert's membrane and the visceral yolk sac from the embryo. Remove the embryos from the uterus. Place the dissected embryos into a separate Petri dish with sterile 1X Krebs buffer.</li>
	<li>Dissect the brain under a stereomicroscope. To dissect the brain, first cut off the head of the embryo. Fix the head by piercing fine forceps through the head (eye level). Use another pair of forceps to carefully remove the skin and skull. Use forceps to carefully lift the brain out and transfer it into a Petri dish with sterile 1X Krebs buffer. It is very important that the integrity of the whole brain is maintained during dissection, since damages to the brain tissue will create problems (such as tissue shredding) during sectioning on the vibratome.</li>
	<li>Wash the brains once in 4 % low-melting point (LMP) agarose. Embed 2-3 brains at a time in fresh 4 % LMP agarose. Place the embedding dishes on ice as even as possible. Use a Pasteur pipette with a fire-polished round tip to lift the brains until the bottom of the agarose is solidified. The brains should settle in a flat position horizontal to the bottom of the agarose block.</li>
	<li>After the agarose has fully solidified (after approximately 3 min), trim the agarose surrounding the brains and glue the agarose block onto the specimen stage of the vibratome. When gluing the blocks, make sure that the ventral side of the brain is parallel to the platform, since the brains should be cut in a horizontal section plane.</li>
</ol>
3. Vibratome sectioning
<ol>
	<li>Use a razor blade for sectioning. To obtain intact slices it is very important to maintain the temperature at 4°C during the sectioning.</li>
	<li>Section 300 μm thick horizontal slices at a frequency of 50 Hz, blade amplitude of 1.1 mm and a speed of 25 mm/sec.</li>
	<li>Use the fine paintbrush to push the slice in a mini perforated spoon to collect the brain slices and transfer them into a dish with sterile ice-cold 1X Krebs buffer. Choose the slice that contains ventral midbrain tissue (see Figure 1). In an E12.5 mouse brain there is just one 300 μm horizontal slice that contains ventral midbrain tissue including dopaminergic neurons.</li>
</ol>
4. Slice culture
Steps 4.2-4.5 should be carried out under sterile conditions.
<ol>
	<li>Transfer the brain slices onto a Millicell cell culture membrane insert in a six-well plate with 1x Krebs buffer (see point 1.2.5). To transfer the slice use the mini perforated spoon (Fine Science Tools) and a fine paintbrush. Up to 3 slices can be placed on one membrane.</li>
	<li>Transfer the membrane with the slice to the six-well plate with culture medium (see point 1.2.5). The top of the membrane should not be covered by medium. The brain slice receives medium from below and air from above.</li>
	<li>Place the six-well plate in a incubator with 5 % CO2 at 37 °C. It is very important that the slices are placed in the incubator within 2 hours after the initial step of the dissection. A more extended preparation time can result in poor survival of the slices.</li>
	<li>Slices can be maintained in vitro up to 3 days. Change 50% of the culture medium on the 2nd day.</li>
</ol>
5. Time-lapse imaging
<ol>
	<li>Let the slices recover in the incubator for 4-5 hours before starting time-lapse imaging.</li>
	<li>For time-lapse imaging, keep the slices on the membrane insert and transfer the insert into a 35 mm Ibidi μ-dish (the bottom of the dish consists of material with high optical quality).</li>
	<li>Add 1 mL of culture medium plus 1.5 μL ascorbic acid (200 mM) to the dish. Ascorbic acid protects the slices against phototoxicity.</li>
	<li>Incubate slices in an environmental chamber at 37 °C with 5 % CO2 during time-lapse imaging.</li>
</ol>
6. Representative Results
Figure 1 illustrates the preparation of organotypic slice cultures from E12.5 mouse brain. Figure 2 shows horizontal organotypic slices (acute and after several days in culture) obtained form E12.5 mouse brain. For comparison, frozen brain sections at the equivalent developmental stages are shown. Midbrain dopaminergic neurons are visualized with immunohistochemistry for tyrosine hydroxylase (TH). Midbrain dopaminergic neurons project to targets in the forebrain. These projections start to form at E12.5 and extend towards the forebrain during the subsequent days. We consider the development of forebrain projections as a good indication for the normal development of dopaminergic neurons in culture. In the horizontal slices midbrain dopaminergic neurons extend projections towards their proper forebrain target area. After 3 DIV (days in vitro) or when the forebrain target areas are damaged, aberrant projections extend towards the dorsal midbrain. Examples of coronal slices of E12.5 midbrain are shown in Figure 3. Dopaminergic neurons visualized with immunostaining for TH extend aberrant projections to the dorsal midbrain. Figure 4 shows an analysis of proliferating, necrotic and apoptotic cells in the organotypic slice cultures. BrdU immunostaining to visualize proliferating cells demonstrates that cells proliferate under culture conditions after 1 DIV. Proliferation is reduced after 4 DIV. After 3 DIV, many cells in the ventral midbrain undergo necrosis (propidium iodide staining) and apotosis (immunostaining for cleaved caspase-3). Figure 5 shows migratory paths of YFP-labeled neurons monitored in a time-lapse imaging experiment in an acute slice.
<img alt="Brain mapping diagram; vibratome sectioning method; slice culture setup; research keywords." src="/files/ftp_upload/3350/3350fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Schematic illustrating the preparation of organotypic slice cultures. 300 μm horizontal brain slices are prepared by sectioning an E12.5 brain using a vibratome. (A) Schematic sagittal view of an E12.5 mouse brain. Levels of sections are indicated. The area containing dopaminergic neurons is depicted in pink, projections are indicated in blue. (B) Schematics of the three slices that can be obtained from dorsal to ventral and that contain both forebrain (Fb) and midbrain (Mb). Note that only one slice contains dopaminergic neurons (slice b). The appropriate slice can be identified based on the position of the ventricles and the continuity of midbrain and forebrain tissue (arrows, compare section b with section a and c). The area containing dopaminergic neurons is indicated in pink, the area containing dopaminergic precursors is depicted in green, blue arrows indicate developing projections. (C) The slice containing dopaminergic neurons is cultured on membrane inserts. Abbreviations: vMb, ventral midbrain; dMb, dorsal midbrain; Hyp, hypothalamus; Hb, hindbrain.
<img alt="Organotypic slice culture and frozen section diagrams show TH immunostaining for neuronal projections." src="/files/ftp_upload/3350/3350fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Projections of midbrain dopaminergic neurons in organotypic slice cultures are dependent on the integrity of the forebrain. Immunohistochemistry for tyrosine hydroxylase (TH) to label dopaminergic neurons. (A) Acute slice (0 DIV) showing the normal location of dopaminergic neurons in the ventral midbrain. The white arrowhead indicates the area that is shown in higher magnification in the inset. Projections to the forebrain have not yet developed. After 1 DIV, projections to the forebrain start to form. Projections extend into the forebrain at 2-3 DIV. White arrowheads indicate the location of the cell bodies shown in higher magnification in the insets (white frame). Yellow arrows highlight the normal projections in intact slices shown in higher magnification in the insets (yellow frame). Aberrant projections develop in slices with damaged forebrain. Red arrows indicate the aberrant projections to the dorsal midbrain shown in higher magnification in the insets (red frame). Damage is indicated with red asterisks. After 3 DIV, most slices (n=5/7) had aberrant projections towards the dorsal midbrain. (B) TH immunostaining on horizontal frozen brain sections at different developmental stages to show the development of dopaminergic projections in vivo. White arrowheads indicate the location of the cell bodies shown in higher magnification in the insets (white frame). Yellow arrows highlight the position of the projections shown in higher magnification in the insets (yellow frame).The level of the frozen sections was chosen to closely match the level of the organotypic slice cultures. Note that a single frozen sections (12 μm) does not represent the entire organotypic slice (300 μm). Therefore, projections shown at E13.5 and E14.5 were observed on sections 120 μm more ventral than the section containing the cell bodies (yellow asterisks).
<img alt="TH immunostaining diagram; 3-stage DIV neural development; fluorescent microscopy." src="/files/ftp_upload/3350/3350fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Midbrain coronal slice cultures stained for tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. (A-C) Slices after 1, 2 or 3 DIV. Dopaminergic neurons develop aberrant projections towards the dorsal midbrain (red arrows). Arrowheads show the location of the dopaminergic cell bodies.
<img alt="Cell proliferation, necrosis, apoptosis in vitro; BrdU, Propidium iodide, cleaved caspase staining, analysis." src="/files/ftp_upload/3350/3350fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. Cell proliferation and cell viability in midbrain organotypic slice cultures. (A) Proliferating cells were labeled by the addition of BrdU (50 ng/mL, Sigma) to the culture medium for 18 h. Slices were subsequently immunostained for BrdU. After 1 DIV, BrdU labeled cells are located in the ventricular zones (red arrows). After 4 DIV, proliferating cells are more scattered and a distinct ventricular zone is no longer maintained. (B) Necrotic cells were labeled by addition of propidium iodide (1μg/μL, Sigma) to the culture medium for 2 hrs and visualized by fluorescence microscopy. After 1 DIV, the ventral midbrain (vMb) is not necrotic, but many propidium iodide labeled cells can be seen in the dorsal midbrain and forebrain. After 3 DIV the cell viability decreases in the ventral midbrain. Scale bar: 500 μm (C) Immunostaing for cleaved caspase-3 to visualize apoptotic cells. At 1 or 2 DIV, only few dopaminergic neurons (TH) are apoptotic. After 3 DIV, dopaminergic neurons start to undergo apoptosis. Panels in the middle and on the right are higher magnifications of the boxed area in the left panels. The higher magnifications images are maximum intensity projections of z-stacks of 14-16 frames. Images were taken every 0.5 μm with an Zeiss Apotome set-up.
<img alt="Fluorescence microscopy diagram of YFP in acute brain slice E12.5; time-lapse visualization." src="/files/ftp_upload/3350/3350fig5.jpg" data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Figure 5. Migratory route of YFP-labeled neurons in an acute slice. (A) Horizontal slice used for time lapse imaging of YFP-labeled neurons. The slice was incubated for 5 hrs prior to imaging. Cells were labeled using an inducible Cre/loxP system6. ShhCreER mice 8 and ROSAloxP-STOP-loxP-EYFP reporter mice 9 were used. Recombination of the ROSA reporter allele (and EYFP expression) is induced in cells that express CreER (Shh-expressing cells), but only upon administration of Tamoxifen (Sigma). In this example, Tamoxifen (3 mg/40 g body weight) was administered to pregnant mice at E8.5. This experimental set-up results primarily in the labeling of precursors of dopaminergic neurons and their descendants in the ventral midbrain10,11 . Scale bar: 500 μm. (B) Tracing the migratory route of YFP-labeled neurons in an acute slice. Time-lapse images of YFP-labeled fate mapped neurons were acquired every 30 min for a total time of 5 hrs 30 min on a Zeiss Axio-Observer microscope (objective EC PlnN 10x /0.3). Slices were incubated in an environmental chamber (incubator XLS1 Pecon) at 37 °C and supplied with 5 % CO2 during the imaging. The initial position of the cells is marked with red arrows; migration positions are marked with red asterisks.

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We have nothing to disclose.

The organotypic slice culture method presented here provides a system for the short-term in vitro analysis of developing dopaminergic neurons and their migratory and projection routes in the embryonic ventral midbrain. We found that there are a number of critical steps in the protocol that should be carefully attended to in order to obtain slices that allow the normal development of ventral midbrain dopaminergic neurons. The most critical step is the dissection of the embryonic brain, which has to be both fast a...

<table><tbody><tr><td colspan="4">Table of specific reagents and equipment</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>Glucose 30%</td><td>Sigma-Aldrich</td><td>G7528-250</td><td></td></tr><tr><td>Horse Serum</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>DMEM (4,5g/L Glc., with L-Gln, Na Pyr, NaHCO3) </td><td>Sigma-Aldrich</td><td>D6429-500</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x</td><td>Sigma-Aldrich</td><td>P4333-20</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma-Aldrich</td><td>A4403</td><td>prepare 200mM stock and store at -20&amp;#x00B0;C</td></tr><tr><td>UltraPure LMP agarose</td><td>Invitrogen</td><td>15517-022</td><td></td></tr><tr><td>Millicel inserts</td><td>EMD Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>&amp;mu;-dish 35 mm, low</td><td>Ibidi</td><td>80136</td><td></td></tr><tr><td>Vibratome</td><td>Microm International</td><td>HM 650V</td><td></td></tr><tr><td>Razor Blade</td><td>Plano GmbH</td><td>121-6</td><td></td></tr><tr><td>Histoacryl glue</td><td>BRAU9381104</td><td>Braun Aesculap</td><td></td></tr><tr><td>Perforated Spoon Dia diameter 15 mm </td><td>Fine Science Tools</td><td>10370 -18</td><td></td></tr><tr><td>Forceps 5 Dumoxel</td><td>Fine Science Tools</td><td>11252 - 30</td><td></td></tr><tr><td colspan="4">Antibodies used for immunostainings:</td></tr><tr><td>Rabbit anti-tyrosine hydroxylase </td><td>EMD Millipore</td><td>AB152</td><td>Dilution 1:500</td></tr><tr><td>Mouse anti-tyrosine hydroxylase </td><td>EMD Millipore</td><td>MAB318</td><td>Dilution 1:500</td></tr><tr><td>Mouse anti-BrdU </td><td>BD Biosciences</td><td>555627</td><td>Dilution 1:200</td></tr><tr><td>Rabbit anti-cleaved caspase 3</td><td>Cell Signaling Technology</td><td>9661</td><td>Dilution 1:200 </td></tr><tr><td>Donkey anti-rabbit IgG-Alexa 488 </td><td>Invitrogen</td><td>A21206</td><td>Dilution 1:500</td></tr><tr><td>Donkey anti-mouse IgG-Cy3</td><td>Jackson ImmunoResearch</td><td>715-165-150</td><td>Dilution 1:200</td></tr></tbody></table>

organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro

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.

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 ...

The overall goal of this procedure is to generate organotypic slices from the E 12.5 neuron embryonic midbrain for the analysis of developing dopaminergic neurons. This is accomplished by first dissecting the embryonic brain. Then the brain is embedded into low melting aeros.The third step is to section the brain slices using a vibrator. The final step is to choose the right brain slice for culturing and time-lapse imaging. Ultimately, time-lapse imaging and immunofluorescence microscopy are used to show the early steps of dopaminergic neurons, migratory behavior and differentiation.This method can help to answer key questions in the neurodevelopmental field, such as following neuron and migratory behavioral during the developmental ventral brain. One day before the dissection, prepare one X Krebs buffer and the culture medium according to the accompanied manuscript about two hours before dissection. Prepare 100 milliliters of 4%low melting aros in Krebs buffer by microwaving the solution until the aros is completely dissolved.Then place the aros in a 45 degree Celsius water bath. Next, fill up the Vibram buffer tray with ice cold Krebs buffer. Turn the cooling element on and keep it at four degrees Celsius.After that, fix a razor blade in the blade carrier. Prepare a scalpel, a fine paintbrush, and a mini perforated spoon for picking up the slices in the vibrator area for later sectioning. Then add Krebs buffer to the sterile Petri dishes and keep them on ice.In the dissection area, prepare three large sterile Petri dishes for dissection and two small sterile Petri dishes for embedding a pair of small scissors, two pairs of fine forceps, a mini perforated spoon, a glass pasture pipet fire polished with a round closed tip and one liter of Krebs buffer on ice. Wipe all the dissection tools with 70%ethanol. Next, add culture medium to the wells of a six Well plate and incubate it at 37 degrees Celsius for two hours.Next, add 1.5 milliliters of Krebs buffer with penicillin streptomycin to each well of another six well plate under sterile conditions. Place milli cell membrane inserts in the wells. Place the six well plate next to the vibrato so that the brain slices can be transferred onto the filter membranes.Immediately after sectioning, sacrifice a pregnant female mouse with embryos at stage E 12.5 and remove the uterus by lifting it up with a pair of forceps. Then use another pair of forceps to tear the myometrium away from the uterus. After that, place the uterus in ice cold Krebs buffer under the microscope.Use a pair of fine forceps to separate the muscular wall of the uterus, riker's membrane, and the visceral yolk sack from the embryo. Then remove the embryos from the uterus and move the embryo to a separate Petri dish with sterile Krebs buffer. Next, cut the head off each of the embryos.Fix the head by piercing through it at the eye level with a pair of fine forceps. Use another pair of forceps to carefully remove the skin and the skull. Then carefully lift the brain up and transfer it into a Petri dish with sterile Krebs buffer.After that, wash the brains in 4%Low melting point aros. Embed two to three brains at a time in 4%fresh, low melting point aros. Using a Pasteur pipette with fire polished round tip.Then place the embedding dishes on ice as even as possible. Next, lift the brains until the bottom of the aros is solidified, so the brains can settle evenly and be parallel to the bottom of the aros block. When the aros has fully solidified trim the aros surrounding the brains.After that glue, the aros block onto the specimen stage of the vibrator. When gluing the block, make sure that the ventral side of the brain is parallel to the platform. Section 300 micron thick horizontal slices at a frequency of 50 hertz blade amplitude of 1.1 millimeters and a speed of 25 millimeters per second.Collect the brain slices by using the fine paintbrush to push the slice to a mini perforated spoon. Then transfer them to a dish with sterile ice cold Krebs buffer under the microscope. Choose the slice that contains ventral midbrain tissue.At this step, use a mini perforated spoon and a fine paintbrush to transfer the brain slices onto milli cell membrane inserts in a six well plate with Krebs buffer that was prepared earlier. Next, transfer the membranes with the slices to another six. Well plate with culture medium, keep the top of the membranes dry so that the brain slices receive medium from below and air from above.Place the slices in an incubator with 5%carbon dioxide at 37 degrees Celsius. It is very important that the slices are placed in the incubator within two hours after the initial step of the dissection. These slices can be maintained in vitro for three to four days.After four to five hours of recovery. The slices can be used in time-lapse imaging right before imaging. Add 1.5 microliters, 200 millimolar ascorbic acid to the dish to protect the slices against phototoxicity.Incubate the slices in an environmental chamber at 37 degrees Celsius with 5%carbon dioxide during time-lapse imaging. Here is an acute slice showing the normal location of dopaminergic neurons in the ventral midbrain in which the projections to the forebrain have not yet developed. After one day in culture, the projections to the forebrains start to form as indicated by the yellow arrow.White arrowhead indicate the location of the cell bodies shown in higher magnification in the insets. Here is the migratory root of YFP labeled neurons. In an acute horizontal slice, the initial position of the cells is marked with a red arrowhead.After watching this video, you should have a good understanding of how to prepare genotypic slices from the E 12.5 marine embryonic midbrain to obtain viable and intact slices. It is important to perform the dissection of the embryonic brain carefully and after vibrato sectioning to choose the correct brain slice for culturing and TimeLapse imaging.

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Research

JoVE Journal

Neuroscience

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Organotypic Slice Culture of E18 Rat Brains

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons.   They can be used to monitor real-time dopamine release kinetics and protein/mRNA  levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

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

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

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

The video demonstrates the process of isolating and culturing dopamine neurons from the midbrain of mouse embryos. The isolated ventral midbrain floors are dissociated using enzymes. The resulting dopamine neurons are harvested and transferred into micro-islands inside multi-well plates. The cells are incubated with a dopamine neuron medium to maintain the cells at a high density inside the micro-islands.

Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons

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.

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized  protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

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.

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

Learn organotypic slice culture of E12.5 murine embryonic midbrain for studying dopaminergic neuron development in vitro. This method enables time-lapse imaging of neuronal migration and differentiation.

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

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