This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

NOTE: All animal procedures are following the guidelines and have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut.
NOTE: For these experiments constitutive NG2cre18 (JAX#008533) and inducible NG2creER16 (JAX#008538) transgenic mice crossed to Z/EG19 (JAX#003920) or gtRosa26:YFP reporter20 (JAX#006148) lines respectively were used to image NG2 cells and their progeny. To image mature oligodendrocytes, PLPDsRed transgenic mice21 were used. For consistent survival, slices can be isolated from mice up to postnatal day 10 from both the forebrain and cerebellum.
1. Slice Preparation
<ol>
	<li>In a culture hood, place tissue culture inserts in six well plates and add 1 ml slice culture medium (recipe in reagents section). Put the plates in the cell culture incubator at 37 &#176;C with 5% CO2 at least 2 hr prior to dissection.</li>
	<li>Sterilize all tools and tissue chopper with 70% ethanol.</li>
	<li>Bubble dissection buffer (recipe in reagents section) with 95% &#160;O2, 5% CO2 on ice for at least 15 min&#160;prior to the start of dissection.</li>
	<li>Anesthetize mouse pups by placing them on ice for 5-10 min according to approved animal protocols. Ascertain the appropriate depth of anesthesia by confirming that the mice do not respond to tail and toe pinching.</li>
	<li>Decapitate animals using sharp scissors following animal protocols. Quickly remove the skull over the forebrain and cerebellum by first making a sagittal cut followed by lateral incision, using sterilized tools. Cut cranial nerves from the ventral surface of the brain and cerebellum by carefully rolling the tissue to the side.</li>
	<li>Place tissue in a sterile 35 mm dish containing ice-cold oxygenated dissection buffer.</li>
	<li>Using a razor blade, sever the cerebellum and forebrain. Then make a mid-sagittal cut through the forebrain and cerebellum, separating the hemispheres.</li>
	<li>Cut 300 &#956;m-thick coronal and sagittal sections of the forebrain and cerebellum with a manual tissue chopper. NOTE: A manual tissue chopper allows rapid processing from dissection to culture incubation without the need for agarose embedding. A vibratome can also be used as described previously9.</li>
	<li>Transfer separated slices into freshly bubbled cold dissection buffer on ice.</li>
	<li>Use small dissecting or weighing spatulas to separate individual sections and transfer them to preincubated six well dishes with culture inserts.</li>
	<li>Remove any excess dissection buffer from the surface of the culture insert using a disposable transfer pipette or a P 200 pipet tip. Two to three forebrain or three cerebellar slices can be placed on one culture insert. 
	NOTE: For consistent results with forebrain cultures, it is ideal to take slices spanning the anterior one-third of the corpus callosum (Figure 1A) in order to study both gray and white matter regions in the same slice. Each animal usually yields 6 forebrain slices and 6 cerebellum slices.</li>
	<li>Place the six well plates in the incubator and replace the slice medium with 1 ml of slice medium 1 day after slice preparation and then every other day until slice fixation.</li>
	<li>Depending on the experiment, use slices after 5-7 days in culture. Use only slices that become transparent after the first few days and discard those that have uneven opaque regions. NOTE: Opaque regions within slices are visible by eye and appear as a clump of dark cells under phase contrast. After the technique has been mastered, dead opaque slices occur rarely, in less than 1-5% of slices.</li>
</ol>
2. Time-Lapse Imaging of NG2 cell Division and Oligodendrocyte Differentiation
NOTE: To perform time lapse imaging of NG2 cell proliferation and differentiation we use NG2cre:ZEG mice16 which express EGFP in NG2 cells and their progeny (Figure 1C-E). Reporter expression in this line is sufficiently robust to obtain live images of GFP+ cells in slices immediately after sectioning for a time period of at least four weeks. The clearest images are obtained after the initial thinning period of the slice, which occurs over the first 3-5 days in culture.
<ol>
	<li>Throughout the experiment, keep the slices in a cell culture incubator at 37 &#176;C and remove only for brief periods of time (less than 15 min per slice), keeping the lid on the six well plate while imaging.</li>
	<li>Using an inverted fluorescence microscope equipped with a digital camera, capture images at the first time point using a 10X objective. NOTE: First time points will vary by experiment and can be the day when the slices were prepared or any time point after.</li>
	<li>Capture images from multiple regions at time point one in both gray and white matter areas of the slice. Note the region imaged by specific landmarks within the slice and by mapping the image location on a schematic that can be used for subsequent imaging sessions. Useful landmarks can include edges of the slices and/or orientation of white matter tracts. Image four to eight locations on each slice at each time point.</li>
	<li>Capture subsequent images at an interval of 4-6 hr if examining NG2 cell division and oligodendrocyte differentiation, ideally over 5-7 days.</li>
	<li>Capture a final image of all regions and fix the slices immediately. Add 1 ml of fixative (4% PFA with 0.1M l-lysine 0.01 M sodium meta-periodate) to the bottom of the culture insert, then add another 1 ml on top of the slices. Take care not to squirt the fixative directly on the slice as it can detach from the membrane. Fix the tissue for 30 min &#160;and proceed with immunohistochemistry using developmental stage-specific markers as described in section 4.</li>
	<li>Wash slices with 0.2 M sodium phosphate buffer 3 x 10 min. If necessary, store slices at 4 &#176;C in 0.2 M sodium phosphate buffer for 1-3 days before immunostaining but ideally perform staining within 24 hr. Proceed with immunohistochemistry as described below in section 4 to determine the proportion of cells that have differentiated into oligodendrocytes (Figure 2D-F).</li>
</ol>
3. Fate Mapping of NG2 cell Progeny Using Inducible Reporter Transgenic Mice in Slice Cultures
NOTE: To track the fate of NG2 cells from a particular time point in the culture, inducible NG2creER:YFP transgenic mice can be used.
<ol>
	<li>Induce Cre recombination and reporter expression by adding 100 nM 4OHT dissolved in ethanol to the culture medium. NOTE: Reporter positive cells should appear within 1-2 days at an induction efficiency of ~20-25% YFP+NG2+/NG2+ cells and at this time point will all be cells at the NG2 cell stage (Figure 2A-C)</li>
	<li>Fix and wash the slices at different time points after various manipulations (described in sections 2.5 and 2.6).</li>
</ol>
4. Slice Immunohistochemistry
<ol>
	<li>Cut the membranes with the slices attached out of the inserts using a scalpel.</li>
	<li>Transfer slices to individual wells of a 24 well plate containing phosphate buffered saline (PBS) using a set of tweezers, taking care not to disrupt the composition of the slice when picking up the membrane.</li>
	<li>Transfer the slices to a 24 well plate with blocking solution containing 5% normal goat serum (NGS) and 0.1% Triton-X100 in PBS for 1 hr.</li>
	<li>Incubate the slices in primary antibody O/N in 5% NGS in PBS at 4 &#176;C. For identifying cells in the NG2 cell stage, use antibodies to NG2 and PDGFR&#945;. For identifying differentiated oligodendrocytes, use an antibody to the adenomatous polyposis coli antigen (APC; clone CC1).</li>
	<li>On the second day wash slices in PBS 3 x15 min, then incubate in secondary antibodies in 5% NGS in PBS for 1 hr at RT. Wash slices in PBS 3 x15 min and mount on slides. Mount with slices up and membrane facing the slide so that the membrane will not be between the objective and the tissue.</li>
</ol>

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The authors declare that they have no competing financial interests

Myelination in the central nervous system is essential for efficient neuronal communication and axonal survival22. NG2 cells continuously generate myelinating oligodendrocytes into adulthood while maintaining a resident population in most brain regions16,23&#8211;25. Some genetic and molecular mechanisms regulating the differentiation of these cells have been described but much remains to be discovered. Organotypic slice cultures are a convenient tool to investigate these mecha...

Organoytpic slice cultures of the central nervous system have proven to be extremely useful for studying neuron and glial cell biology in a semiintact system1&#8211;4. These cultures are relatively simple to adopt and retain many benefits of primary dissociated cell cultures such as manipulation of the extracellular environment and easy access for repeated long-term live cell imaging and electrophysiological recordings5&#8211;9. In addition, slice cultures maintain 3-dimensional tissue cytoarchitecture, regional neural connectivity, and most major cell types are present in the system. These properties make these cultures a unique and convenient system to investigate single cell behavior and physiology with cellular and environmental interactions.
NG2 cells are a population of glial cells in the mammalian central nervous system that continue to proliferate and generate myelinating oligodendrocytes during embryonic and postnatal development10. They have been extensively studied in dissociated primary cell cultures, and recent development of transgenic mouse lines with NG2 cell-specific expression of fluorescent proteins has facilitated in vivo fate mapping and electrophysiological recordings in acute slice preparations. Even with these studies, little is known about the temporal dynamics of NG2 cell proliferation and oligodendrocyte differentiation. Although dissociated cell culture is widely used for the relative facility in pharmacological and genetic manipulations, it is not suitable for interrogating functional differences of these cells in different brain regions particularly when it is desirable to maintain the context of the cellular microenvironment. Slice cultures provide a simple alternative that is amenable to pharmacological manipulations and have been used to investigate oligodendrocyte myelination11,12, cellular response after lysolecithin (LPC) or antibody induced demyelination13,14, and induction of remyelination via pharmacological treatment15.
A method to investigate and perform live imaging and fixed tissue (or postfixation) analysis of NG2 cell proliferation and oligodendrocyte differentiation in organotypic slice cultures taken from both the forebrain and cerebellum is described. This is a powerful method that can be used to study the cell fate of single NG2 cells after division16 and to discover region- and age-dependent differences in growth factor induced NG2 cell proliferation17. This relatively simple technique is widely accessible to further investigate cell intrinsic and/or environmental mechanisms regulating the physiology of these glial cells and their response to neuronal activity or myelin damage.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Slice Culture Medium&lt;/strong&gt;</td></tr><tr><td>Minimum Essential Medium</td><td>Invitrogen</td><td>11090</td><td>50%</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Invitrogen</td><td>14175</td><td>25%</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td>25mM</td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030</td><td>1mM</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>1mg/L</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A-0278</td><td>0.4mM</td></tr><tr><td>Horse Serum</td><td>Hyclone</td><td>SH30074.03</td><td>25%</td></tr><tr><td colspan="4">Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection Buffer&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>124mM</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P5405</td><td>3.004mM</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td>1.25mM</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td>4.004mM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C7902</td><td>2mM</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td>26mM</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>10mM</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A-0278</td><td>2.0mM</td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>A4036</td><td>0.075mM</td></tr><tr><td colspan="4">Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O&lt;sub&gt;2&lt;/sub&gt; 5%CO&lt;sub&gt;2&lt;/sub&gt; before use</td></tr><tr><td colspan="4">&lt;strong&gt;Other Reagents and Supplies&lt;/strong&gt;</td></tr><tr><td>4-hydroxy tamoxifen (4OHT)</td><td>Sigma-Aldrich</td><td>H7904</td><td>100nM</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>EM Sciences</td><td>19200</td><td>4%</td></tr><tr><td>L-Lysine</td><td>Sigma-Aldrich</td><td>L-6027</td><td>0.1M</td></tr><tr><td>Sodium Metaperiodate</td><td>Sigma-Aldrich</td><td>S-1878</td><td>0.01M</td></tr><tr><td>Rabbit anti NG2 antibody</td><td>Chemicon (Millipore)</td><td>AB5320</td><td>1:500</td></tr><tr><td>Mouse anti CC1 antibody</td><td>Calbiochem (Millipore)</td><td>OP80</td><td>1:200</td></tr><tr><td>Mouse anti Ki67 antibody</td><td>Vision Biosystems</td><td>NCL-Ki67-MM1</td><td>1:300</td></tr><tr><td>Mouse anti NeuN antibody</td><td>Chemicon (Millipore)</td><td>MAB377</td><td>1:500</td></tr><tr><td>Species specific secondary antibodies</td><td>Jackson Immunoresearch</td><td></td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td></td><td></td><td>5%</td></tr><tr><td>Triton X-100</td><td></td><td></td><td>0.10%</td></tr><tr><td>Mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Millicell culture membrane inserts 0.45&amp;mu;m pore size</td><td>Fisher Scientific</td><td>PICM03050</td><td></td></tr><tr><td>Bottle top filters</td><td>Fisher Scientific</td><td>09-740-25E</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt; gas mix</td><td></td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>Sterile six well plates</td><td></td><td></td><td></td></tr><tr><td>Dissecting (hippocampal) or weighing spatulas</td><td></td><td></td><td></td></tr><tr><td>Manual or automatic tissue chopper</td><td></td><td></td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Disposable transfer pipettes</td><td></td><td></td><td></td></tr><tr><td>35mm and 60mm sterile Petri dishes</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Inverted Phase Microscope</td><td></td><td></td><td></td></tr><tr><td>Laminar Flow Hood</td><td></td><td></td><td></td></tr><tr><td>Tissue Culture Incubator</td><td></td><td></td><td></td></tr><tr><td>Inverted Fluorescence Microcope</td><td></td><td></td><td></td></tr></tbody></table>

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.

Examples of representative data are given below that have been obtained using slice cultures from both the forebrain and cerebellum of P8 NG2cre:ZEG, NG2creER:YFP and PLPDsRed transgenic mouse lines. NG2 cells can be imaged over days in forebrain and cerebellum slices (Figure 1B-D, Video 1) and the phenotype of these cells can be determined after fixation and immunostaining with NG2 and CC1 antibodies (Figure 1E). In addition to live imaging, cell proliferation can also be assessed using...

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Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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 ...

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Neuroscience

18.3K Views.  University of Connecticut. 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.

Video: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

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.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunology and Infection

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

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

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

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 ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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