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 mechanisms due to their unique characteristics of maintaining anatomical regions, easy manipulation of the extracellular environment, robust myelination, and the presence of all major cell types. These characteristics facilitate investigation of short and long-term interactions between NG2 cells, oligodendrocytes and axons11,26. In addition, cell transplantation is relatively easy to perform and can be used to investigate region-dependent differences in cell behavior17. Furthermore, pharmacological treatments can be added to the culture medium to investigate molecular mechanisms influencing NG2 cell proliferation and/or differentiation in normal17,27,28 and demyelinated cultures15,29. Finally, it is technically feasible to use slice cultures to perform screens for high throughput analysis of compounds that direct NG2 cells to proliferate or differentiate, possibly even after a demyelinating insult30.
Current methods to investigate oligodendrocyte lineage cells and their interactions with axons in a controlled culture setting include co-cultures with dissociated dorsal root ganglion (DRG) or embryonic cortical neurons and NG2 cells31,32, which were based on original preparations developed to investigate DRG-Schwann cell interactions33. These cultures have been used to investigate fundamental properties of axon and oligodendrocyte lineage cell interactions including neuronal activity-dependent signaling to induce differentiation and myelin production32,34&#8211;36 in addition to other questions such as the dependence of axon diameter and NG2 cell density controlling proliferation and the onset of differentiation37. While these coculture systems are ideal to address such questions, direct correlation and application to the in vivo situation is not always clear. As mentioned previously, organotypic slice cultures provide a unique condition of maintaining many of the local neuronal connections and three dimensional cytoarchitecture that are lost in co-culture preparations of dissociated cells. Furthermore unlike organotypic slice cultures, co-culture systems of dissociated cells often do not include other glial cells and extracellular matrix molecules which have shown to play key roles in the development, maintenance and physiology of NG2 cells, oligodendrocytes, and myelin. With the dramatic growth in the variety of tools available for specific labeling and manipulation of distinct cell populations with viral vectors and transgenic animals, long-term imaging and fate mapping in organotypic slice cultures should prove to be a powerful technique for the investigation of NG2 cell proliferation, differentiation and oligodendrocyte myelination.
Several critical steps should be carried out when performing these experiments to ensure slice survival and data reproducibility. First, in order to generate healthy slices, sectioning and isolation of slices should be performed as quickly as possible and in ice cold dissection buffer. Second, while automated image position acquisition with a motorized stage on the fluorescence microscope can be useful, there are many factors that can disrupt the precise relocation of the regions imaged and manual repeated acquisition is necessary to get consistently reliable images over multiple days. This is particularly necessary if the slices are kept in a cell culture incubator between imaging sessions and not in a stage-top incubator. Third, during slice fixation, staining, and mounting it is extremely important to handle the slices gently as any disruption of the tissue will result in the inability to relocate live imaged regions and cells. There are no specific modifications to traditional methods used for slice culture preparations that are used for studies of neurons and astrocytes.
There are several limitations to the slice culture method that must be considered. Astrocytes, microglia and NG2 cells are known to respond to CNS damage with increased proliferation at the site of injury and the formation of a glial scar. The process of preparing the slices results in some reorganization and an initial reactive glial response from these cells. NG2 cell proliferation rates return to levels similar to those reported in vivo for matched stages of development after several days in culture however the increased proliferation rates and initial reactive phenotype of these cells must be considered when interpreting the data. The presence of horse serum in the culture medium could alter NG2 cell proliferation and should be considered when interpreting the findings of any experiment. In addition to this, while there is spontaneous neuronal activity, sensory input and long range connections between neurons are lacking, which could lead to altered neuronal activity within the slice. Finally, without the use of specific promoter driven viral vectors or transgenic mice, cell type-specific genetic manipulations within slice culture systems is challenging. While these limitations should be considered when designing experiments, slice cultures are a unique system which can be utilized to gain novel insight into questions that cannot be answered with dissociated cell cultures or in the intact animal.

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;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></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</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>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</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></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</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&#956;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% O2 5% CO2 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 immunohistochemical staining for cell proliferation markers such as Ki67 (Figure 1F). These data allow for detailed analysis of the temporal dynamics of oligodendrocyte differentiation after NG2 cell division on an individual cell basis. Fate mapping of NG2 cells can be carried out after induction of cre recombination in slices with 4OHT in NG2creER:YFP mice (Figure 2). YFP reporter-expressing NG2+ cells were found in the cultures two days after addition of 4OHT to the culture medium (Figure 2A). Six days after 4OHT induction, a proportion of the YFP+ cells had differentiated into CC1+ oligodendrocytes (Figure 2B). These data show the feasibility of performing fate mapping of NG2 cells in slices where the extracellular environment can be manipulated to test the effects of pharmacological agents or various insults on the fate of NG2 cells. Different concentrations of 4OHT can be used to achieve different degrees of cre recombination. For example, exposure to 100 nM 4OHT for 2 days causes reporter expression in approximately 20-25% of all NG2 expressing cells while 1&#181;M 4OHT results in approximately 60-70% recombination efficiency. Finally, extensive myelin is present in these cultures and myelinating oligodendrocytes can be imaged in live and fixed slices using PLPDsRed transgenic mice (Figure 3, Video 2).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51835/51835fig1highres.jpg" width="500" /> 
Figure 1.&#160;Slice culture method for long-term imaging of NG2 cell proliferation and differentiation (A-B) Schematic depicting the isolation of forebrain and cerebellum slices (A) and subsequent culturing and time-lapse imaging using slice culture inserts (B). (C) Depiction outlining NG2 cell division and subsequent oligodendrocyte differentiation in NG2cre:ZEG transgenic mice.&#160;(D) Example images acquired from cortical regions of NG2cre:ZEG mice showing a single GFP+ cell (arrows) dividing twice over a period of 122 hr, time depicted in top right corner as hours after the start of the imaging experiment. (E) Fixation and immunostaining with anti-NG2 and CC1 antibodies of the same slice reveals that the imaged dividing cells resulted in three NG2+ cells (arrows). (F) Example image showing two Ki67+NG2+ cells in a cortical slice culture. (G) Example image showing NG2+ cells (arrowheads) and their processes intertwined with NeuN+ neuronal nuclei in a cortical slice culture. Scale Bars 25 &#956;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51835/51835fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51835/51835fig2highres.jpg" width="500" /> 
Figure 2.&#160;Induction of YFP expression to track the fate of NG2 cells in forebrain slice cultures (A-B) Images taken of the corpus callosum region of forebrain slice cultures from NG2creER:YFP mice treated with 4OHT for 24 hr and then left in culture for 2 days (A) or 6 days (B). Slices were fixed and immunostained for NG2 and YFP (A) or CC1 and YFP (B) demonstrating the differentiation of reporter positive NG2 cells into oligodendrocytes while in culture 6 days after cre recombination (arrow). Scale Bars 25 &#956;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51835/51835fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51835/51835fig3highres.jpg" width="500" /> 
Figure 3.&#160;Imaging oligodendrocytes in slice cultures from PLPDsRed transgenic mice. (A) Schematic depicting the isolation, culturing and imaging of cerebellum slices from PLPDsRed transgenic mice. (B) Image taken from a fixed cerebellum slice after 5 days in vitro showing calbindin+ (cyan) purkinje neurons with myelinated myelin basic protein+ (MBP) (green) axons projecting into white matter regions of the slice. Scale Bar 25 &#956;m.&#160;(C) Low magnification images captured from the same region every other day at the times indicated in hours in cerebellum slice cultures from PLPDsRed mice. Scale Bar 100 &#956;m.&#160;(D) Low magnification image taken from a fixed PLPDsRed slice culture showing MBP expression in white matter regions where DsRed+ cells are concentrated. Scale Bar 100 &#956;m.&#160;(E) High magnification image taken from a fixed PLPDsRed slice culture showing single DsRed+ oligodendrocytes with MBP+ processes. Scale Bar 20 &#956;m.&#160;(F) Time-lapse sequence taken from a PLPDsRed cerebellum slice showing relatively stable cell bodies over the 48 hr imaging session, time indicated in upper right in hours. Scale Bar 25 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51835/51835fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1.&#160;Live Imaging of NG2 cell division in a cortical slice culture. Representative time lapse-sequence showing multiple cell divisions in a cortical slice culture taken from an NG2cre:ZEG transgenic mouse. Video displayed at 5 frames per second, montage of images shown in Figure 1. (See &#34;Video_1.mov&#34; under Downloads)
Video 2.&#160;Live imaging of oligodendrocytes in cerebellum slice cultures Representative time lapse-sequence showing small changes in oligodendrocyte morphology (arrow) imaged over 48 hr in a cerebellum slice taken from a PLPDsRed transgenic mouse. Video displayed at 3 frames per second&#160;, montage of images shown in Figure 3. (See &#34;Video_2.mov&#34; under Downloads)

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

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 ...

organotypic-slice-cultures-to-study-oligodendrocyte-dynamics

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.2K 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

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 Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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 ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

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. ...

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 ...