The authors would like to thank James Oesterling for flow cytometry assistance. This research was supported by a grant from the US National Cancer Institute (R01-CA178380, Z.Y.) and a US National Institutes Postdoctoral training grant (5T32CA009035-37, L.W.Y.).

Use of animals in this protocol has been performed in accordance with procedures approved by the Fox Chase Cancer Center Animal Care and Use Committee.
1. Preparation of Instruments, Solutions, and Coverslips
<ol>
	<li>Clean two sets of #5 fine forceps, one set of #7 fine curved forceps, one large surgical scissor, one microdissecting scissor, one spatula and one perforated spoon. Place the instruments in a self-sealing sterilization pouch and autoclave. Keep instruments in pouch until ready for use.</li>
	<li>Prepare a 3% low melting temperature agarose solution.
	<ol>
		<li>Add 1.5 g agarose into 50 ml of sterile Dulbecco&#8242;s Phosphate Buffered Saline (DPBS) then place in microwave and heat until bubbles appear. Swirl flask and reheat until all agarose has dissolved. Mix solution with a stir bar prior to heating if clumps appear.</li>
		<li>Store solution in a 37 &#176;C water bath until needed.</li>
		<li>For long-term storage, store the agarose solution for future use at room temperature or at 4 &#176;C for months. Reheat in microwave to liquefy. For repeated heating of the agarose solution, weigh the flask before heating to maintain its initial weight with warm sterile distilled water.</li>
	</ol>
	</li>
	<li>Prepare the following solutions for papain-based cell dissociation as previously described2:
	<ol>
		<li>A papain solution with papain (100 U/ml), cysteine (0.2 mg/ml) and DNase (250 U/ml) in sterile phenol red-containing DPBS.</li>
		<li>An ovomucoid solution with bovine serum albumin (BSA; 8 mg/ml), soybean trypsin inhibitor (8 mg/ml)) and DNase (250 U/ml) in sterile phenol red-containing DPBS. 
		Filter sterilize both solutions and adjust pH to return original color of phenol red-containing DPBS.</li>
	</ol>
	</li>
	<li>Prepare cell culture media (NB/B27): supplement basal media with 1 mM sodium pyruvate, 2 mM L-glutamine, 1% penicillin-streptomycin (P/S) and B27 supplement. Filter sterilize and protect from light. Equilibrate media at 37 &#176;C and 5% CO2 before use.</li>
	<li>Prepare FACS buffer: 5% fetal bovine serum (FBS) in DPBS. Add 2.5 ml FBS to 47.5 ml DPBS.</li>
	<li>Prepare poly-D-lysine (PDL)-coated coverslips.
	<ol>
		<li>Autoclave new glass coverslips.</li>
		<li>Coat the coverslips with PDL (100 &#956;g/ml in sterile distilled water) and incubate for 2 hr at 37 &#176;C or room temperature overnight.</li>
		<li>Wash coverslips with sterile distilled water. Wash once with&#160;NB/B27 media. Let coverslips dry completely in tissue culture hood prior to use.</li>
	</ol>
	</li>
</ol>
2. Dissection and Preparation of Tissue Slices
<ol>
	<li>On the day of dissection, wipe down the dissection area with 70% ethanol and cover with an absorbent pad.</li>
	<li>Remove dissection tools from the sterilization pouch. Add ice cold, sterile DPBS/1% P/S to a small Petri dish and place on ice.</li>
	<li>Decapitate P4 Math1-GFP/Nestin-CFP mouse pups with large surgical scissors then hold the head down with fine curved forceps. Remove the skin and gently peel away the skull with #5 fine forceps then scoop out the brain from the skull using a spatula and transfer it to the Petri dish containing DPBS/1% P/S.
	<ol>
		<li>Using #5 fine forceps, carefully separate the cerebellum from the rest of the brain. Be sure to dissect one pup at a time as quickly as possible.</li>
		<li>Collect a minimum of 8 cerebella in order to obtain enough NEPs for molecular and functional analyses.</li>
	</ol>
	</li>
	<li>Fill an embedding mold measuring 2 x 2 x 2 cm with 3% low melting temperature agarose solution. Ensure that the temperature of agarose is no more than 37 &#176;C.</li>
	<li>Remove excess liquid from cerebellum by dabbing gently on a laboratory tissue. Place cerebellum into mold in a vertical position. Place the mold on ice to allow it to solidify quickly. Use ~4 cerebella per block.</li>
	<li>Obtain living tissue sections with a vibrating blade vibratome. Trim cerebella-containing agarose block with a razor blade. Glue the agarose block to the vibratome plate with cerebella oriented in a vertical position.</li>
	<li>Fill the tray of the vibratome with ice-cold sterile DPBS/1% P/S and place ice underneath.
	<ol>
		<li>Cut 600 &#956;m sections through the whole length of cerebellum. Collect sections from the ice tray using a perforated spoon. Place sections in a Petri dish filled with DPBS/1% P/S on ice.</li>
	</ol>
	</li>
</ol>
3. Microdissection and Cell Dissociation
<ol>
	<li>Place the Petri dish containing tissue slices under a fluorescent dissecting microscope.
	<ol>
		<li>Carefully separate the EGL from the rest of the cerebellar section using fine forceps by dissecting in between the CFP+ molecular layer and the GFP+ EGL (refer to dotted line in Figure 1). Be careful to not include any part of the molecular layer, which will result in contamination of Nestin-expressing Bergmann glia.</li>
		<li>Complete this step as quickly as possible and keep the tissue on ice to avoid cell death.</li>
		<li>Remove the agarose surrounding the tissue before proceeding to the next step.</li>
	</ol>
	</li>
	<li>Digest the microdissected EGL tissue using a papain-based protocol as previously described2 to obtain a single cell suspension.
	<ol>
		<li>Re-suspend cells in FACS buffer for collection of CFP-positive cells via FACS.</li>
	</ol>
	</li>
</ol>
4. Cell Sorting and Plating
<ol>
	<li>Sort cells for CFP fluorescence using a sterile, high speed flow cytometer containing an appropriate CFP filter and collect cells in FACS buffer on ice. Obtain approximately 100,000 NEPs from each P4 cerebellum.</li>
	<li>Centrifuge cells at 300 x g for 5 min. Use cells immediately for molecular analysis or culture for further experimentation.</li>
	<li>To culture cells, re-suspend the cells in pre-warmed NB/B27 media and plate on PDL-coated coverslips as previously described2.</li>
</ol>

<ol>
	<li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Wechsler-Reya, R. J., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+neuronal+precursor+proliferation+in+the+cerebellum+by+Sonic+Hedgehog.">Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.</a> Neuron. 22, 103-114 (1999).</li><li>Dahmane, N., Ruizi Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126, 3089-3100 (1999).</li><li>Riobo, N. A., Manning, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+signal+transduction+employed+by+vertebrate+Hedgehogs.">Pathways of signal transduction employed by vertebrate Hedgehogs.</a> Biochem J. 403, 369-379 (2007).</li><li>Dahmane, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sonic+Hedgehog-Gli+pathway+regulates+dorsal+brain+growth+and+tumorigenesis.">The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.</a> Development. 128, 5201-5212 (2001).</li><li>Marino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+developmental+mechanisms+out+of+control.">Medulloblastoma: developmental mechanisms out of control.</a> Trends Mol Med. 11, 17-22 (2005).</li><li>Yang, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nat Neurosci. 16, 1737-1744 (2013).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Sutter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+stem+cells+act+as+medulloblastoma-initiating+cells+in+a+mouse+model+and+a+neural+stem+cell+signature+characterizes+a+subset+of+human+medulloblastomas.">Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas.</a> Oncogene. 29, 1845-1856 (2010).</li><li>Sotelo, C., Alvarado-Mallart, R. M., Frain, M., Vernet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+plasticity+of+adult+Bergmann+fibers+is+associated+with+radial+migration+of+grafted+Purkinje+cells.">Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.</a> J Neurosci. 14, 124-133 (1994).</li><li>Lumpkin, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr Patterns. 3, 389-395 (2003).</li><li>Encinas, J. M., Vaahtokari, A., Enikolopov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoxetine+targets+early+progenitor+cells+in+the+adult+brain.">Fluoxetine targets early progenitor cells in the adult brain.</a> Proc Natl Acad Sci U S A. 103, 8233-8238 (2006).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+astroglial+morphology+and+proliferation+in+vitro.">Neuronal regulation of astroglial morphology and proliferation in vitro.</a> J Cell Biol. 100, 384-396 (1985).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), (2009).</li><li>Emmert-Buck, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Espina, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nat Protoc. 1, 586-603 (2006).</li></ol>

The authors have nothing to disclose.

The microdissection method described here is the first procedure able to isolate a pure population of living cells for use in molecular and functional analyses. As demonstrated by Li et al.9, NEPs obtained by this method can be cultured and incorporated into various experiments and because of their high purity, can also be used for microarray analysis.
It is very important to take time to become proficient with this microdissection technique. NEPs are located in the deep region of the EGL in very close proximity to the molecular layer, in which Nestin-expressing Bergmann glia could contaminate the NEP cell isolation and compromise purity. Microdissection should therefore be done conservatively to make sure contaminating cells in adjacent tissue are not included. The major advantage of the microdissection technique described here is the ability to obtain living cells that can be cultured for further experimentation. Because of this, it is crucial that all steps during this procedure be done quickly and that tissue be kept on ice as much as possible. Also, all instruments and reagents must be sterile. Following these steps will reduce cell death and cell culture contamination. In addition, mounting multiple cerebella per agarose block will aid in reducing the procedure time.
This technique is not limited to isolation of NEPs and can be used to collect cells from other areas of the cerebellum, such as glial cells from the molecular layer and neural stem cells from the white matter. Beside the cerebellum, microdissection can be used with other tissues that contain recognizable regions, such as the hippocampus, for example. The use of fluorescent transgenes, as utilized here, can aid in creating borders or identify regions for dissection, which can be especially helpful in tissues that lack easily identifiable areas and would otherwise not be able to be microdissected. This technique could therefore aid in cell purifications in a wide range of research areas and applications.

The cerebellum is comprised of multiple cell layers, each containing distinct cell types. During development, the EGL contains proliferating granule neuron precursors (GNPs) while the molecular layer and Purkinje layer contain Bergmann glia and Purkinje neurons, respectively. Deep within the cerebellum lies the white matter, which contains neural stem cells (NSCs) and oligodendrocytes1.
Sonic hedgehog (Shh) plays a critical role in regulating cerebellar development and in particular, it promotes the proliferation of GNPs via binding to its receptor Patched (Ptc), a negative regulator of Shh signaling2-4. Aberrant activation of Shh signaling generates medulloblastoma (MB), the most common malignant brain tumor in children5,6.
Ptc mutant MB transgenic mouse models are powerful tools for the study of MB and develop tumors resembling human MB7,8. Using these mice, it was discovered that GNPs are the cell of origin for hedgehog-type MB7. Furthermore, in addition to GNPs, we have recently identified a unique population of neuronal progenitors within the EGL of the developing cerebellum that can also give rise to MB. These cells express high levels of the type VI intermediate filament protein, Nestin, and are termed NEPs9. NEPs are located within the deep part of the EGL and exist transiently only during neonatal cerebellar development. They are committed to the granule cell lineage as GNPs but are distinct as they are quiescent and do not express Math1, a well-established marker for conventional GNPs10. In addition, NEPs give rise to MB more efficiently than GNPs after activation of the Shh signaling pathway9, which makes them a novel origin for MB tumorigenesis.
We previously isolated NEPs from the cerebellar EGL at postnatal day 4 (P4) by the microdissection technique described here9. Microdissection via direct microscopic visualization of the tissue allows for specific dissection of the cerebellar EGL. This is necessary as Nestin is also expressed by NSCs in the cerebellar white matter and by Bergmann glia in the molecular layer1,7,11,12 and it was crucial that these cells not be included, as they would confound analysis. Cells isolated from microdissected tissue can be used immediately for molecular analysis or they can be cultured for further applications.
To aid in specifically microdissecting the EGL and to further purify NEPs, Math1-GFP (green fluorescent protein) mice were crossed with Nestin-CFP (cyan fluorescent protein) mice. The transcription factor Math1 is specifically expressed by GNPs and GFP expression is clearly visible in the EGL9,13. Nestin-CFP mice express a nuclear form of CFP and can be easily visualized in the molecular layer9,14. Together, GFP and CFP expression create boundaries for microdissection of the EGL (see Figure 1). CFP-positive NEPs were then isolated by FACS, following enzymatic dissociation of the dissected EGLs.
Currently, the most well utilized method to isolate cells from the EGL is Percoll gradient centrifugation of whole cerebellar tissue15,16. This method, however, is unable to completely exclude Nestin+ cell populations from the molecular layer and white matter and therefore cannot be used for the study of NEPs. Laser capture microdissection, which uses the transfer of laser energy to remove cells of interest, is another method used to isolate specific cells within a heterogeneous tissue17,18 but captured cells can only be used for recovery of DNA, RNA and protein and are not able to be cultured.
This protocol provides a way to specifically isolate an alive, pure population of NEPs from the EGL. This technique can also be applied to different tissue types that have either recognizable anatomical architecture or fluorescently labeled cells/regions. Therefore, the major advantage of incorporating this novel microdissection technique into cell isolation protocols is that cells can be isolated from specific tissue regions to eliminate contaminating neighboring tissue and the cells can be collected immediately for analysis or cultured for other applications.

<table border="0" cellpadding="0" cellspacing="0" width="572">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of distinct cell populations from the developing cerebellum by microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

As shown in Figure 1A, cerebellar slices were prepared from P4 Math1-GFP/Nestin-CFP mice, in which the cerebellar EGL was dominated by GFP-expressing GNPs and the molecular layer was enriched by CFP-positive glial cells. To isolate NEPs that are located in the deep part of the EGL, slices were microdissected between the EGL and the molecular layer (along dashed line; Figure 1A). Dissected EGLs (Figure 1B) were then collected for enzymatic dissociation followed by FACS.
As expected, a majority (more than 85%) of cells in the dissected EGL were conventional GNPs, which were positive for GFP (Figure 2A). NEPs (CFP+) account for only around 5% of the EGL cell population. Almost none of the cells were double-positive for GFP and CFP based on the FACS analysis. CFP+ cells purified by FACS, were cultured in vitro to examine the differentiation potential. As shown in Figure 2B, NEPs exclusively gave rise to &#946;-tubulin+ neurons after 4 days in culture, suggesting NEPs are lineage-restricted neuronal progenitors.
By this microdissection protocol, NEPs and GNPs were also purified from the EGL of Ptc deficient cerebellum. Upon intracranial transplantation, NEPs develop MBs more rapidly compared with GNPs, indicating that NEPs are particularly susceptible to oncogenic transformation9.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52034/52034fig1highres.jpg" width="500" /> 
Figure 1. Identification of the region for EGL microdissection. Fluorescent images from P4 Math1-GFP/Nestin-CFP animals. (A) GFP expression is located in the EGL, while the majority of CFP expression is located in the molecular layer. Microdissection was done along the yellow dotted line. (B) EGLs were collected for tissue dissociation. This figure has been modified from Li et al9.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52034/52034fig2highres.jpg" width="500" /> 
Figure 2. NEPs represent a small population of cells in the EGL and are lineage-restricted neuronal progenitors. (A) Flow cytometry analysis of CFP+ cells isolated from microdissected EGL. (B) NEPs were cultured under differentiation conditions for 4 days and stained for &#946;-tubulin (red) and counterstained with DAPI (blue). This figure has been modified from Li et al.9

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

isolation-distinct-cell-populations-from-developing-cerebellum

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.5K Views.  Temple University Health System. Nestin-expressing progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Video: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

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Chapters in this video

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum