This work was funded by the Defense Advanced Research Projects Agency (DARPA) and the Army Research Office (ARO), award number W911NF-10-1-006.

1. Sampling setup and preparation
<ol>
	<li>Supplement Earle's balanced salt solution (EBSS) with sodium bicarbonate (0.44 g per 100 mL EBSS) and glucose (0.884 g per 100 mL EBSS)9. Treat the EBSS with DEPC (0.1 mL per 100 mL EBSS) for at least 12 hours at 37 &deg;C in an autoclavable bottle or flask with a screw top.</li>
	<li>Place 5 &frac34;" glass Pasteur pipets into a 1000 mL glass beaker, and place artist's brushes, with bristles facing upward, into a 100 mL glass graduated cylinder. Add sufficient DEPC solution (0.1 mL per 100 mL ddH2O) to cover the pipets and brushes, respectively. Cover the beaker and graduated cylinder with aluminum foil, and incubate for at least 12 hours at 37 &deg;C.</li>
	<li>After 12-hr incubation with DEPC, heat all DEPC-treated materials to a temperature &gt;100 &deg;C for at least 15 minutes. Remove EBSS and cool to room temperature prior to storage at 4 &deg;C. Pour ddH2O away from the beaker and graduated cylinder; continue to incubate at &gt;100 &deg;C until materials are dry. Remove, cool to room temperature, and store for later use.</li>
	<li>On the day of the experiment, treat all work surfaces with RNaseZap.</li>
	<li>Place and maintain EBSS in a water bath set at 40 &deg;C. To buffer and oxygenate the EBSS, infiltrate with 95%/5% O2/CO2 by placing gas tubing into a RNase-free Pasteur pipet and inserting the pipet into the EBSS. Cover the opening of the EBSS container.</li>
	<li>Set up a Stoelting tissue slicer for use in serial sectioning of the brain tissue. Treat the top and bottom surfaces of the tissue slicer with RNaseZap. Attach a rectangular piece of Whatman #4 filter paper to the plexi-glass surface of the tissue chopper. Attach a double-edge razor blade to the chopper arm. Set the micrometer to zero, and adjust the minimum height of the chopper arm to allow the razor blade to cut the filter paper, but not the plexi-glass plate.</li>
	<li>Gather all additional materials needed for the microdissection, including RNase-free 60x15 mm cell culture dishes, Harris Uni-Core tissue corers, 200 &mu;L RNase-free micropipette tips for affixing to gas tubing for EBSS oxygenation of coronal brain sections during microsample punching, curved scissors, dry ice, and tubes for tissue collection. Place tubes on dry ice.</li>
</ol>
2. Brain removal and coronal sectioning
<ol>
	<li>Place the mouse into a DecapiCone and decapitate using a rodent guillotine. Remove the head from the DecapiCone and place onto a flat surface. Using a scalpel, cut the skin down the midline of the skull starting approximately 3 mm caudal to the nostril. Laterally peel the skin flaps back to expose the cranium and the eye sockets.</li>
	<li>Using curved scissors, cut both optic nerves, severing their connection to the eye. Open the cranial cavity by carefully breaking the occipital and interparietal bones and removing the dorsal parietal and frontal bones. Using a bone rongeur, break the nasal and lacrymal bones to expose the olfactory bulbs. Use curved scissors to cut the olfactory bulbs away from the rostral brain. Remove the whole brain with hindbrain intact. Place the brain into the top of a 60 x 15 mm, RNase-free Cellstar cell culture dish. Transfer to the tissue sampling work surface.</li>
	<li>Use an RNase-free pipet to conservatively moisten the filter paper to the attached tissue chopper with EBSS. Wet the brain tissue with EBSS.</li>
	<li>Transfer the brain to the tissue chopper and set up appropriate orientation. Note: The brain should be positioned with the ventral surface resting on the filter paper. The midline (interhemispheric fissure) should be perpendicular to the chopper blade, and the rostral edge of the brain should abut the blade.</li>
	<li>Use a DEPC-treated artist's brush to hold the brain stationary by applying gentle pressure to the dorsal surface of the brain. While holding the brain stationary, lift the blade holder lever up about 4-5 cm above the dorsal tissue surface and hold it in this position. Remove the brush from the brain surface, and dial 750 &mu;m using the micrometer. Allow the lever to drop, chopping the tissue and creating a 750 &mu;m coronal section.</li>
	<li>While the chopper blade remains stationary, use a rolling motion with the artist's brush to gently swipe the tissue section away from the blade surface. Immediately place the tissue section into the oxygenated EBSS in the petri dish. A second investigator can start on step 3.1.</li>
	<li>Repeat steps 2.5 and 2.6 until serial brain sections have been obtained across the entire rostro-caudal plane.</li>
</ol>
3. Brain region microdissection
<ol>
	<li>Adjust lighting to ensure that sample illumination will not be obstructed during tissue microdissection. Adjust the position and flow of 95%/5% mix of O2/CO2 to maintain a buffered, oxygenated environment for the tissue during microdissection.</li>
	<li>Identify the coronal brain section that includes the region of interest. Orient the brain section flat on the bottom of the petri dish using an artist's brush. Select the tissue corer with appropriate diameter based on Table 1 or other references. While holding the coronal tissue section in place with the artist's brush, bring the cutting tip of the tissue corer down over the surface of the tissue section.</li>
	<li>While applying pressure, press the tissue corer tip through the tissue section and down firmly onto the bottom of the petri dish. Use a rolling circular motion to ensure that the region of interest has been dissociated from the surrounding tissue. Carefully lift the tissue corer away from the tissue section and liberate the micropunch, allowing it to float in the EBSS.</li>
	<li>Using an artist's brush, immediately deposit the tissue micropunch into a -80 &deg;C-stable storage tube kept on dry ice. The tissue should readily adhere to the side of the tube. Return the microsample-containing tube to the dry ice.</li>
	<li>Repeat steps 3.2 - 3.4 for each brain region of interest.</li>
</ol>
4. Tissue archival
<ol>
	<li>Once the microdissection is complete, use the VisionMate scanner system to record the respective bar codes for each sample-containing TrakMates tube. Once the 96-slot TrakMates tube rack has been filled, quickly scan the rack and immediately place the tissue samples into a -80 &deg;C freezer until RNA extraction.</li>
</ol>
5. Total RNA extraction
<ol>
	<li>Prepare a RNase-free workspace using RNaseZap. Prepare a sufficient amount of magnetic Bead Mix and Lysis/Binding Solution as described within the MagMAX-96 Total RNA Isolation Protocol10.</li>
	<li>Remove tissue samples from the -80 &deg;C freezer and place the tubes directly onto dry ice.</li>
	<li>Add 100 &mu;L of the Lysis/Binding Solution to a sample-containing TrakMates tube. Attach a nuclease-free 0.5 ml tube-sized pestle to the pellet mixer and homogenize the tissue. Using a low-retention nuclease-free pipet tip, transfer the homogenized sample into a well on a round-bottom 96-well tissue culture plate. Immediately add 60 &mu;L of 100% isopropanol to the sample using a low retention nuclease-free pipet tip, and mix by pipeting up and down 3-4 times.</li>
	<li>Add 20 &mu;L of the Bead Mix to the sample using a low retention nuclease-free pipet tip. Mix by pipeting up and down 3-4 times.</li>
	<li>Repeat steps 5.3-5.4 for all tissue samples.</li>
	<li>Follow the MagMAX-96 Total RNA Isolation Protocol steps starting at III.A.2 to complete the total RNA extraction. NOTE: Other magnetic bead-enabled total RNA isolation kits are commercially available; however, they were not examined in this study. Please refer to the manufacturer's kit manuals for the details of total RNA isolation using the respective products.</li>
	<li>Measure the purity and concentration of resulting total RNA using a NanoDrop 3300 spectrophotometer or other small-volume spectrophotometer/fluorometer technologies.</li>
</ol>
6. Representative Results:
<table border="1">
	<tbody>
		<tr>
			<td>Brain Region/Nucleus</td>
			<td>Tissue Corer Diameter (mm)</td>
			<td>Total RNA (ng/&mu;L)</td>
			<td>260/280 Ratio</td>
		</tr>
		<tr>
			<td>Frontal Cortex</td>
			<td>2.0</td>
			<td>27.03</td>
			<td>1.99</td>
		</tr>
		<tr>
			<td>Nucleus Accumbens</td>
			<td>1.0</td>
			<td>6.26</td>
			<td>2.77</td>
		</tr>
		<tr>
			<td>Striatum</td>
			<td>1.5</td>
			<td>27.94</td>
			<td>2.19</td>
		</tr>
		<tr>
			<td>Cingulate Cortex</td>
			<td>1.5</td>
			<td>29.05</td>
			<td>2.07</td>
		</tr>
		<tr>
			<td>Hypothalamus - Paraventricular Nucleus / Suprachiasmatic Nucleus (PVN/SCN)</td>
			<td>2.0</td>
			<td>40.34</td>
			<td>2.03</td>
		</tr>
		<tr>
			<td>Thalamus</td>
			<td>2.0</td>
			<td>34.40</td>
			<td>2.00</td>
		</tr>
		<tr>
			<td>Hypothalamus - Dorsomedial Nucleus / Ventromedial Nucleus (DMH/VMH)</td>
			<td>2.0</td>
			<td>44.83</td>
			<td>2.05</td>
		</tr>
		<tr>
			<td>Amygdala</td>
			<td>1.0</td>
			<td>10.06</td>
			<td>1.89</td>
		</tr>
		<tr>
			<td>Hippocampus - Ca 2/3</td>
			<td>1.5</td>
			<td>7.31</td>
			<td>2.32</td>
		</tr>
		<tr>
			<td>Hippocampus - Ca1/Dentate Gyrus</td>
			<td>1.5</td>
			<td>5.20</td>
			<td>2.24</td>
		</tr>
		<tr>
			<td>Periaqueductal Grey</td>
			<td>1.0</td>
			<td>48.21</td>
			<td>2.02</td>
		</tr>
		<tr>
			<td>Dorsal Raphe Nucleus</td>
			<td>1.0</td>
			<td>6.56</td>
			<td>2.18</td>
		</tr>
		<tr>
			<td>Reticular Formation</td>
			<td>2.0</td>
			<td>35.35</td>
			<td>2.27</td>
		</tr>
	</tbody>
</table>
Table 1. Enumeration of microdissected mouse brain regions with respective optimal tissue corer diameters, representative total RNA yield and purity. The 13 microdissected brain regions, listed in rostro-caudal order, vary in size and orientation. The PVN/SCN micropunch includes the anteromedial paraventricular nucleus, portions of the periventricular nucleus, the sub-paraventricular zone and other parvocellular neurons. The thalamus micropunch includes anterior thalamic nuclei situated in the same coronal plane as the PVN/SCN micropunch region. The DMH/VMH micropunch includes accessory magnocellular nuclei and other parvocellular neurons situated between the dorsomedial nucleus and ventromedial nucleus. The amygdala sample includes both the central and basolateral subregions. The RNA yield and 260/280 ratios are single values obtained from respective microdissection samples to demonstrate readings obtained from each brain region.

<ol>
	<li>Pickrell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+mechanisms+underlying+human+gene+expression+variation+with+RNA+sequencing.">Understanding mechanisms underlying human gene expression variation with RNA sequencing.</a> Nature. 464, 768-772 (2010).</li><li>Costa, V., Angelini, C., De Feis, I., Ciccodicola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+the+complexity+of+transcriptomes+with+RNA-Seq.">Uncovering the complexity of transcriptomes with RNA-Seq.</a> J. Biomed. Biotechnol. 853916, (2010).</li><li>Nagalakshmi, U., Waern, K., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+method+for+comprehensive+transcriptome+analysis.">RNA-Seq: a method for comprehensive transcriptome analysis.</a> Curr. Protoc. Mol. Biol. Chapter 4, 11-13 (2010).</li><li>Anglicheau, D., Muthukumar, T., Suthanthiran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+small+RNAs+with+big+effects.">MicroRNAs: small RNAs with big effects.</a> Transplantation. 90, 105-112 (2010).</li><li>Forrest, A. R., Carninci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+genome+transcriptome+analysis.">Whole genome transcriptome analysis.</a> RNA. Biol. 6, 107-112 (2009).</li><li>Twine, N. A., Janitz, K., Wilkins, M. R., Janitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+transcriptome+sequencing+reveals+gene+expression+and+splicing+differences+in+brain+regions+affected+by+Alzheimer's+disease.">Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer's disease.</a> PLoS One. 6, e16266-e16266 (2011).</li><li>Paulsen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+individual+hypothalamic+nuclei+from+single+animals+using+laser+capture+microdissection+and+microarrays.">Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays.</a> J. Neurosci. Methods. 177, 87-93 (2009).</li><li>Winrow, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+anatomical+isolation+of+functional+sleep+circuits+exhibits+distinctive+regional+and+circadian+gene+transcriptional+profiles.">Refined anatomical isolation of functional sleep circuits exhibits distinctive regional and circadian gene transcriptional profiles.</a> Brain. Res. 1271, 1-17 (2009).</li><li>Atkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+integration+of+glutamatergic+signals+by+little+SAAS+in+novel+suprachiasmatic+circuits.">Circadian integration of glutamatergic signals by little SAAS in novel suprachiasmatic circuits.</a> PLoS One. 5, e12612-e12612 (2010).</li><li>Technologies, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MagMAX-96 Total RNA Isolation Kit Instruction Manual. , .</li></ol>

No conflicts of interest declared.

There are many critical steps in this protocol that warrant careful attention and consideration. Selection of the appropriate tissue corer diameter must be determined for removing each respective region of interest. While the tissue corer diameters presented in Table 1 are suitable for collection of the listed brain regions, each investigator should determine the appropriate-sized tissue corer for optimal collection of their respective tissue(s) of interest. Secondly, the coronal section thickness reported in this met...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Isotemp 102 water bath</td>
			<td>Fisher Scientific</td>
			<td>15-460-3Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase Zap</td>
			<td>Ambion</td>
			<td>AM9782</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lab rotator (orbital shaker)</td>
			<td>Thermo Scientific</td>
			<td>2314 &#x2013; 1CEQ</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pellet mixer</td>
			<td>VWR</td>
			<td>47747-370</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase-free 0.5 mL pestle</td>
			<td>VWR</td>
			<td>KT749521-0590</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma Life Science</td>
			<td>D5758-50ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Double-edge razor blades</td>
			<td>Stoelting </td>
			<td>51427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stoelting tissue slicer with manual micrometer</td>
			<td>Stoelting </td>
			<td>51425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2/0 Round artist's brush</td>
			<td>Princeton Art &amp; Brush</td>
			<td>4350R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (0.75 mm)</td>
			<td>Ted Pella</td>
			<td>15072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (1.0 mm)</td>
			<td>Ted Pella</td>
			<td>15099</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (1.5 mm)</td>
			<td>Ted Pella</td>
			<td>15075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (2.0 mm)</td>
			<td>Ted Pella</td>
			<td>15076</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Guillotine</td>
			<td>Braintree Scientific</td>
			<td>RG-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DecapiCones</td>
			<td>Braintree Scientific</td>
			<td>MDC-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HyClone Earle's balanced salt solution (EBSS)</td>
			<td>Thermo Scientific</td>
			<td>SH30029.02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 mL pipets</td>
			<td>Corning</td>
			<td>4489</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>epT.I.P.S. LoRetention Dualfilter pipet tips, 20 &#x2013; 300 &mu;L</td>
			<td>Eppendorf</td>
			<td>0030 077.636</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TrakMates screw top tubes with caps</td>
			<td>Thermo Scientific</td>
			<td>3741</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VisionMate wireless 2D barcode reader</td>
			<td>Thermo Scientific</td>
			<td>3122MTX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VisionMate SR single rack 2D barcode reader</td>
			<td>Thermo Scientific</td>
			<td>3115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1-200 &mu;L universal fit pipette tips</td>
			<td>Corning</td>
			<td>4864</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Cellstar cell culture dishes</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 96-well polystyrene Cellstar tissue culture plate, U-bottom</td>
			<td>USA Scientific</td>
			<td>5665-0180</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>#4 Filter paper</td>
			<td>Whatman</td>
			<td>1004 150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silicone bulb-type safety pipette filler</td>
			<td>Fisherbrand</td>
			<td>13-681-102A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NanoDrop 3300 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-3300</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

non-laser capture microscopy approach for the microdissection of discrete mouse brain regions for total rna isolation and downstream next-generation sequencing and gene expression profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Watch this Scientific Journal Video about Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencin

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

non-laser-capture-microscopy-approach-for-microdissection-discrete

Research

JoVE Journal

Neuroscience

14.7K Views.  Northwestern University. RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Video: Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

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 ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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 ...

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 ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...