This work was supported by the NIH, DA043982 and DA046196.

Animals used in this study were treated in an ethical and humane manner as set forth by Indiana University&#8217;s Institutional Animal Care and Use Committee (IACUC) and National Institutes of Health (NIH) guidelines.
NOTE: All tools and surfaces should be washed with an appropriate solvent to remove nucleases18 before starting any work.
1. Storing brains
<ol>
	<li>Quickly remove the brains from euthanized adult CD1 wildtype mice weighing approximately 30 g using a conventional approach13 and flash-freeze for 60 seconds in either liquid nitrogen or isopentane pre-chilled with dry ice.</li>
	<li>Store frozen brains at -80 &#176;C in aluminum foil or conical tubes until use.</li>
</ol>
2. Preparing the brain matrix
<ol>
	<li>Twenty-four hours before dissecting tissue, place a clean brain matrix (see Table of Materials) on a stack of thawed freezer packs. Sandwich the sides of the matrix between two freezer packs making sure to leave approximately 0.5 cm between the bottom of the razor slots and the top of the gel packs (Figure 1A). Place aluminum foil on the ends to aid in cooling. 
	NOTE: When purchasing a brain matrix, buy one that is large enough to encase the entire volume of the brain to be dissected.</li>
	<li>Place the box containing the brain matrix and freezer packs in a -20 &#176;C freezer with the top ajar overnight.</li>
</ol>
3. Setting up a frozen glass plate
NOTE: The purpose of this setup is to prepare a frozen surface on which to dissect brain sections.
<ol>
	<li>Place ice in an insulated box up to approximately 5 cm from the top. Then place a 2.5 cm layer of dry ice on top of the ice and cover with black plastic sheeting to aid in visualizing the sample (Figure 1B, Figure 1C).</li>
	<li>Place a glass plate (should just fit in the opening of the box) on top of the plastic and place dry ice on top of the plate in the far corners (Figure 1C).</li>
	<li>Take the frozen brain matrix from the freezer and insert the brain to be cut cortex-side up. Allow it to equilibrate to the temperature in the box for 10 min. Keep the lid on the box during this time.</li>
	<li>Adjust the brain&#8217;s position in the matrix with cold forceps so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block (Figure 1D). This will help ensure symmetric sections for easier dissection. Touch tips of forceps to dry ice briefly to chill before adjusting the brain.</li>
	<li>Once the brain is in position, place a chilled razor blade near its center and press the blade approximately 1 mm into the tissue. Add chilled blades to the rostral and caudal ends to help hold the brain in place (Figure 2A and Figure 2B).</li>
	<li>Starting on the rostral end, add blades one at a time, placing them into the slots and pressing them gently down into the tissue approximately 1 mm (Figure 2B). Continue to add blades at 1 mm intervals working towards the caudal end.</li>
	<li>Make sure the brain does not shift during this time. Line up blades horizontally and vertically (Figure 2B). In order to cut sections into 0.5 mm widths, low profile microtome blades (see Table of Materials) may be used. Place these between the larger razor blades (Figure 2C).</li>
	<li>Once blades are in place, press down on top of the group with fingers, palm or some other flat surface (Figure 2C, Figure 2D). Rock the group of blades slowly from side to side to move them through the tissue. 
	NOTE: This may take some time (1&#8211;2 min) and requires patience. There should be resistance to the blades moving through the tissue. Easy entry means the brain is thawing and the block should be cooled with dry ice.</li>
	<li>When all blades have reached the bottom of the slots, grasp each side of the group of blades and work free of the matrix by rocking back and forth. 
	NOTE: Exercise caution to avoid cutting oneself on the sharp edges of the blades.</li>
	<li>Once the group is free, place the stack, rostral side up, on the glass plate (Figure 2E). Place dry ice next to and/or on top of the stack to further freeze the samples for easier separation.</li>
	<li>Place the stack with sharp edges down on the glass plate and separate blades by shifting the stack between thumbs and fingers. The blades should separate from each other with sections attached.</li>
	<li>Line up sections on the glass plate from rostral to caudal (Figure 2F).</li>
	<li>Separate tissue from the blades by flexing blades between fingers, or by separating with a second cold razor (Figures 2G,2H,2I).</li>
</ol>
4. Dissecting sections
<ol>
	<li>Open the Allen Mouse Brain Atlas or another reference and find landmarks necessary to identify regions of interest. Some obvious landmarks include the anterior commissure, the corpus callosum, the lateral ventricles, and the hippocampus (Figure 3).</li>
	<li>Flip the section to be cut with chilled forceps and make sure the region about to be collected is consistent throughout the section. During harvesting, consult the reference atlas often to make sure the correct ROI is obtained. 
	NOTE: A magnifying glass or dissecting microscope is often helpful in this process. Good lighting is also essential. Low wattage LED lamps or a cool lamp (see Table of Materials) can be used for this purpose.</li>
	<li>With a clean scalpel or punch, cut into the section (Figure 4). Prechill each tool before cutting by touching it briefly to dry ice. Push the blade gently but firmly into the tissue, rocking it back and forth to make the cut. Do not push too hard or the tissue will fracture. 
	NOTE: Tools will warm over time. Slightly warmed blades can be helpful in making clean cuts and can limit fracturing, but be careful to avoid thawing of tissue. Periodically chill tools on dry ice.</li>
	<li>Once an ROI is harvested, place it into labeled, prechilled 1.5 mL tubes. Store harvested tissues at -80 &#176;C until needed.</li>
	<li>Process frozen tissue collected in this manner by adding cold RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% CHAPS and protein inhibitor cocktail (see Table of Materials)) for protein extraction, or a guanidinium-containing solvent (see Table of Materials) for RNA extraction to the frozen sample and immediately homogenize using either a glass Dounce or mechanical homogenizer (see Table of Materials). In this way, protease and nuclease inhibitors are in place as the sample warms to protect protein and nucleic acids from degradation.</li>
</ol>

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F., 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=Cannabinoid+Exposure+via+Lactation+in+Rats+Disrupts+Perinatal+Programming+of+the+Gamma-Aminobutyric+Acid+Trajectory+and+Select+Early-Life+Behaviors.">Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors.</a> Biological Psychiatry. , (2019).</li><li>Choi, D. W., Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+glutamate+neurotoxicity+in+hypoxic-ischemic+neuronal+death.">The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.</a> Annual Review of Neuroscience. 13 (1), 171-182 (1990).</li><li>Guhaniyogi, J., Brewer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mRNA+stability+in+mammalian+cells.">Regulation of mRNA stability in mammalian cells.</a> Gene. 265 (1-2), 11-23 (2001).</li><li>Sampaio-Silva, F., Magalh&#227;es, T., Carvalho, F., Dinis-Oliveira, R. 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The authors have nothing to disclose.

This work describes a technique to isolate small, specific regions of brain while limiting degradation of nucleic acid and protein. Damage to brain tissues happens quickly once an organism dies. This is partially due to a rapid buildup of extracellular glutamate and the resultant excitotoxicity that occurs21. Messenger RNA is particularly vulnerable to degradation22,23. Breakdown of protein and nucleic acid is greatly reduced at low temperatures as evidenced by a recent article describing biological activity in mammoth cells frozen in permafrost 28,000 years ago24.
With the method described herein, brains are snap frozen and tools and tissues are kept below freezing until nucleic acid or protein extraction is started. Frozen ROIs are then homogenized in buffer containing protease and nuclease inhibitors, protecting target molecules from degradation at higher temperatures.
In this process, it is important to identify clear landmarks to pinpoint ROIs within the sections. Fiber tracts of the anterior commissure and corpus callosum work well for this purpose, as do the ventricles. Changes in the shape of the hippocampus as one moves anterior to posterior can also be used. Depending on how well the brain is aligned in the matrix, landmarks may be skewed, or missing. Caution should be taken before collecting ROIs from sections like this as contaminating regions may end up in the sample. Before collection, an ROI should be checked on both sides of the section to make sure it is consistent throughout the section. Dissection of ROIs is limited to well defined areas of the brain that are greater than approximately 1 mm in the x-y dimension.
When dissecting ROIs, there is a need to keep tissues frozen without making them too brittle. Tissue that is too cold will fracture when pressure is applied with a punch or scalpel. The region of interest is often hard to identify from other fragments when this happens. One way to mitigate this is to briefly move the sections to a warmer area of the plate (away from dry ice). It is also important to keep the glass plate clean. Once a section is processed, the plate should be scraped clean with a razor blade before moving on to the next. Over time, water will condense onto the dissection plate from the air which may cover or otherwise interfere with sample recognition. Cleaning the plate is done by scraping the sharp edge of a razor blade across the surface of the glass and wiping up the collected frost and debris. Small ROIs become indistinguishable from unwanted brain fragments so it is very important to maintain a clean working area.
Many labs use commercial products25 to protect RNA in harvested tissues. While labile molecules are well preserved this way, one drawback is that the morphology of the tissue is radically changed. As a result, it may be difficult to find the regions of interest using reference maps. Dissecting ROIs from snap-frozen brains before using these products may therefore be a better option.
Although we have focused on coronal slices in this work, this method should work well for horizonal and sagittal sections. Maps for these orientations are generally lower in resolution as compared to those for coronal sections. However, this is changing rapidly with initiatives from groups like the Allen Brain Institute. Another consideration is the age of the animal at harvest. The morphology of young animal brains is very different from that of the adult and will require a specialized atlas and a different size brain matrix. Resources like the Developing Mouse Brain Atlas26 are essential in collecting accurate brain samples from younger mice.
This work describes a process that can be used to harvest small regions from within the rodent brain. The morphology of sections is well preserved with frozen slicing, and as the tissues are kept frozen from harvest to extraction, the quality of nucleic acid and protein is high. This process has the advantage over the dissection of freshly collected tissue as sample brains from a large group of animals can be quickly collected and stored. Regions within brains can then be identified using landmarks identified in common brain atlases and collected at a careful pace without losing target molecules. This method may be a good option for some labs as the tools and regents needed are inexpensive and readily available.

This work illustrates the dissection of frozen brain regions for extraction of high-quality nucleic acid or protein using a reference, such as the Allen Mouse Brain Atlas1, as a guide. In this technique, brains are flash-frozen and stored at -80 &#176;C for later sectioning and dissection while being maintained in a frozen condition. This process allows the researcher to harvest a large number of brains in one session and later dissect them for an accurate collection of multiple brain regions.
The accurate collection of brain regions of interest (ROIs) is often required when answering questions related to gene and protein expression. While pharmacology, electrophysiology and optogenetics can be used on wildtype or genetically modified rodents to help elucidate molecular changes underpinning observed behaviors2,3,4, the measurement of induced changes in transcriptomes and proteomes is often used to support these findings. Techniques such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blotting, RNAseq5, MAPSeq6 and HPLC7 are robust and relatively low in cost, allowing many labs to study induced molecular changes within small brain regions2,4,5,6.
There are several ways to extract and purify nucleic acid or protein from brain regions8,9,10,11,12. Many labs harvest brain regions by chilling and cutting brains on ice at the time of harvest9,13. While this approach can result in high quality nucleic acid and protein, it is somewhat time-limited as degradation within the microenvironment of the tissue may take place at these temperatures. This may be particularly true when attempting to dissect a large number of animals or ROIs in one sitting. Keeping samples frozen helps maintain labile target molecules while providing the researcher time to carefully compare landmarks on both sides of each section in the effort to collect relatively pure samples. Laser capture is another way to collect tissue for RNA or protein analysis from brain areas10. This procedure is superior to mechanical dissection in that very small and irregularly shaped ROIs can be identified and isolated. However, laser capture is limited by the use of expensive equipment and reagents, is time consuming and may also be more susceptible to sample degradation.
Micropunch dissection on frozen tissues is not new. Early papers by Miklos Palkovits and others describe the basic techniques in detail14,15. This presentation largely follows the original work, with some improvements to facilitate efficiency and decrease the expense of the equipment needed. For instance, brain sections are made in a frozen brain block rather than on a cryostat. This produces thicker sections which reduces the number of sections needed to collect ROI samples. This method also dissects samples on a frozen glass plate that sits on dry ice within an insulated box. This produces a sub-freezing stage at the bench on which to work. Sections dissected in this way are easily manipulatable, allowing the researcher to compare both sides of each section with a reference in order to limit contamination from regions outside the desired ROI.
Advantages of this protocol are that 1) the brain is kept in a frozen condition throughout the process, which helps preserve protein and nucleic acid and gives the researcher time to carefully harvest ROIs, and 2) the reagents required are inexpensive and are found in most molecular biology labs. In this process, whole brains are sectioned to 0.5&#8211;1.0 mm in a brain matrix and placed on a frozen glass plate that is continuously chilled with dry ice. Landmarks found in the Allen Brain Atlas1 or other brain atlases16,17 are used to identify regions of interest, which are then dissected using either a cold punch or scalpel. Because the tissue is never thawed, regions harvested in this manner provide high quality RNA and protein for downstream analyses.

<table><tbody><tr><td>0.5 mm Mouse coronal brain matrice</td><td>Braintree Scientific</td><td>BS-SS 505C</td><td>Cutting block</td></tr><tr><td>0.5 mm Rat coronal brain matrice</td><td>Braintree Scientific</td><td>BS-SS 705C</td><td>Cutting block</td></tr><tr><td>1.0 mm Biopsy Punch with plunger</td><td>Electron Microscopy Sciences</td><td>69031-01</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Dot Scientific</td><td>229443</td><td>For storing frozen ROIs</td></tr><tr><td>1.5 mm Biopsy Punch with plunger</td><td>Electron Microscopy Sciences</td><td>69031-02</td><td></td></tr><tr><td>2.0 mm Biopsy Punch with plunger</td><td>Electron Microscopy Sciences</td><td>69031-03</td><td></td></tr><tr><td>4-12% NuPage gel</td><td>Invitrogen</td><td>NPO323BOX</td><td>protein gradient gel</td></tr><tr><td>Bioanalyzer System</td><td>Agilent</td><td>2100</td><td>RNA analysis system</td></tr><tr><td>Dounce tissue grinder</td><td>Millipore Sigma</td><td>&lt;br/&gt; D8938</td><td>Glass tissue homogenizer</td></tr><tr><td>Dry Ice</td><td></td><td></td><td></td></tr><tr><td>Fiber-Lite</td><td>Dolan-Jenner Industries Inc.</td><td>Model 180</td><td>Cool lamp</td></tr><tr><td>Glass plates</td><td>LabRepCo</td><td>11074010</td><td></td></tr><tr><td>HALT</td><td>ThermoFisher</td><td>78440</td><td>protease inhibitor cocktail</td></tr><tr><td>Low profile blades</td><td>Sakura Finetek USA Inc.</td><td>4689</td><td></td></tr><tr><td>mouse anti-actin antibody</td><td>Developmental Studies Hybridoma Bank</td><td>JLA20</td><td>Antibody</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>2000C</td><td>Used in initial RNA purity analysis</td></tr><tr><td>No. 15 surgical blade</td><td>Surgical Design Inc</td><td>17467673</td><td></td></tr><tr><td>Odyssey Blocking buffer</td><td>LiCor Biosciences</td><td>927-40000</td><td>Western blocking reagent</td></tr><tr><td>Omni Tissue Master 125</td><td>VWR</td><td>10046-866</td><td>Tissue homogenizer</td></tr><tr><td>rabbit anti-KCC2 antibody</td><td>Cell Signaling Technology</td><td>94725S</td><td>Antibody</td></tr><tr><td>RNA Plus Micro Kit</td><td>Qiagen</td><td>73034</td><td>Used to extract RNA from small tissue samples</td></tr><tr><td>RNaseZap</td><td>Life Technologies</td><td>AM9780</td><td></td></tr><tr><td>Scalpel handle</td><td>Excelta Corp.</td><td>16050103</td><td></td></tr><tr><td>Standard razor blades</td><td>American Line</td><td>66-0362</td><td></td></tr><tr><td>TRIzol Reagent</td><td>ThermoFisher Scientific</td><td>15596026</td><td>Used to extract RNA from tissue</td></tr></tbody></table>

collection of frozen rodent brain regions for downstream analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

In order to validate this method, the medial prefrontal cortex was collected from adult CD1 wildtype male mice and RNA and protein were extracted and characterized. RNA was analyzed by capillary electrophoresis. Degraded RNA displays a loss in the intensity of the 28S and 18S ribosomal bands and also shows degradation products as a smear between 25 and 200 nucleotides (Figure 5A, sample 1). High quality RNA shows distinct ribosomal bands with little to no signal in the lower molecular weight region (Figure 5A, sample 2). RNA integrity numbers (RINs) are also generated in this analysis. RINs above 7 indicate good quality RNA19. RNA isolated from samples using the frozen dissection method display strong ribosomal banding and high RIN values comparing very favorably with RNA prepared from freshly harvested tissue (Figure 5B).
One advantage of this method is that a large number of brains can be quickly harvested and stored in one sitting to be carefully dissected at a later time. ROIs can also be stored without the tissues being thawed. With this method, a researcher can collect a large and diverse collection of ROIs from which high-quality nucleic acid or protein may be obtained. To confirm this point, RNA was extracted from the dissected mPFC that had been stored over several weeks (harvested brains had been stored for several months). All samples produced high quality RNA with distinct ribosomal banding and RINs above 8 (Figure 5C).
Western blot analysis was used to confirm protein integrity of frozen dissected mPFC as previously described20. Western blots display reduced signal of high molecular weight targets when protein is degraded. The degradation products also show up as a smear at lower molecular weights within the column. For this analysis protein was collected, transferred to nitrocellulose, and probed with antibodies against a high molecular weight target, KCC2, and the lower molecular weight protein, actin (Figure 5D). In all cases, bands were sharp and distinct with no discernable breakdown products for either KCC2 or actin. These experiments confirm that the dissection of ROIs using this method enables the extraction of high-quality RNA and protein.
<img alt="Cryosectioning process; foam container setup; brain tissue preparation; labeled diagram; sagittal sinus; transverse sinus." class="xfigimg" src="/files/ftp_upload/60474/60474fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Preparing frozen work surfaces. (A) A brain block is placed on a stack of gel packs within an insulated box and is then sandwiched between two additional gel packs. Aluminum foil is packed at each end of the block to facilitate cooling during sectioning. The box is then cooled overnight at -20 °C. (B) A glass plate is matched for size with the opening of an insulated box. A shipping box can be used for this purpose. Ice is added to the box until it is approximately 5 cm from the top. The box is then frozen overnight at -20 °C. (B, C) Just before collecting tissue, a uniform layer of dry ice is placed on top of the ice at a depth of approximately 2.5 cm. A sheet of black plastic (to aid in visualization) is placed over the dry ice followed by the glass plate. Dry ice is placed in the far corners, which cools the air just above the plate. Tools can be cooled by laying them on the plate, or by briefly touching them to the dry ice. (D) To ensure symmetric sections, the brain should be positioned with cold forceps so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block (white arrows). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60474/60474fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cryostat sectioning process; step-by-step tissue preparation using microtome, gloves, slides, ice bath." class="xfigimg" src="/files/ftp_upload/60474/60474fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Sectioning brain using a brain block. (A) The orientation of the brain is anchored by seating conventional single edge razor blades into the cortex at a depth of approximately 1 mm. Placing one in the middle and one at each end helps to hold the brain in position. (B) Add blades one at a time until all slots are filled. (C) Low profile blades can be inserted between conventional blades when 0.5 mm sections are desired (red arrow). (C, D) The group of blades is pushed into the frozen tissue with moderate downward pressure, using either a flat instrument or the palm or fingers of the hand. Blades can be slowly rocked back and forth to help move them through the tissue. (E) Sections are removed by grasping the sides of the razor blades and using a rocking motion and firm upward pressure (Caution: Be careful to avoid sharp ends of the blades). Lift the group out of the block and place it anterior-side up on the frozen glass plate. Place dry ice adjacent to blades to chill sections completely before separating. (F) Separate blades and lay sections out in order. (G, H) Sections can be removed from blades by flexing blade slightly with fingers in the direction of the arrows and pushing the section off with a second cold blade. (I) Some sections may require removal by slightly warming the razor blade between the fingers and thumbs and then quickly slicing off the frozen section with a second cold blade. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60474/60474fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Brain section histology diagram; regions labeled for anatomical study and structure identification." class="xfigimg" src="/files/ftp_upload/60474/60474fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Determining ROIs using landmarks and the Allen Mouse Brain Atlas. In this example, frozen brain sections are on the left with corresponding plates from the Allan Mouse Brain Atlas on the right. ROIs (red dotted outlines) are identified by comparing visible landmarks (black shapes) to the Allen Mouse Brain Atlas. Landmarks include: fa and cc, corpus callosum, aco, anterior commissur, VL, lateral ventricle, V3, third venticle. Example ROIs: Pl/Il, prelimbic and infralimbic cortex, ACB, nucleus accumbens, MOs, motor cortex, CP, caudoputamen, HPF, hippocampus/dentate gyrus. Coronal atlas images on the right credit: Allen Institute. Images are from plates 40, 44, 55 and 71 (top to bottom) and were obtained from https://mouse.brain-map.org/experiment/thumbnails/100048576?image_type=atlas. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60474/60474fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tissue dissection process, experimental setup with scalpels, examines sample precision cutting." class="xfigimg" src="/files/ftp_upload/60474/60474fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Dissecting ROIs. (A) Sections are handled using forceps that are chilled periodically on dry ice. Both sides of each section should be compared with the corresponding atlas images before dissection. (B) ROIs are removed from brain sections with cold scalpel or (C) biopsy punch. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60474/60474fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="RNA analysis gel, electrophoresis results, showing 28S/18S bands, degraded RNA, KCC2, and Actin protein." class="xfigimg" src="/files/ftp_upload/60474/60474fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Analysis of RNA and protein using frozen brain dissection method. (A) Example of poor versus good quality RNA. Electrophoretic separation of sample 1 displays degraded RNA with weak 28S and 18S bands and degradation products between 25 and 200 nucleotides (nt). Sample 2 shows good quality RNA with strong 28S and 18S bands and little signal at low molecular weights. RNA integrity numbers reflect this difference with sample 1, RIN = 2.5 and sample 2, RIN = 8.6. (B) Comparison of RNA extracted from frozen versus freshly dissected medial prefrontal cortex of mice. Frozen samples were kept at -80 °C for several months before dissection. Fresh samples were processed immediately following harvest. RIN values from samples collected using both methods are high, with strong 18S and 28S bands indicating that nucleic acid has been preserved. (C, D) mPFC was dissected from frozen brains and RNA or protein was extracted. (C) RNA analysis shows good quality RNA was obtained for all twelve samples with high RINs and little degradation product observed. (D) Western blot analysis was done on total protein and was probed for the presence of the high molecular weight KCC2 ion channel (red bands, MW = 130 kDa). The KCC2 band is clearly visible with no lower molecular weight breakdown products observed. The housekeeping gene, Actin (green bands, MW = 42 kDa) is also shown. mPFC samples from 12 brains are labeled 1-12. WB stands for whole brain control. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60474/60474fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

collection-of-frozen-rodent-brain-regions-for-downstream-analyses

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

19.6K Views.  Indiana University. This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

Video: Collection of Frozen Rodent Brain Regions for Downstream Analyses

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Biology

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicine

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.

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.

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

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

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 ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Cerebellar Regional Dissection for Molecular Analysis

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies indicate that distinct cerebellar regions contribute to these different functions. Molecular studies examining regional cerebellar differences are lagging as they are mostly done on whole cerebellar extracts thereby masking any distinctions across specific cerebellar regions. Here we describe a technique to reproducibly and quickly dissect four different cerebellar regions: the deep cerebellar nuclei (DCN), anterior and posterior vermal cerebellar cortex, and the cerebellar cortex of the hemispheres. Dissecting out these distinct regions allows for the exploration of molecular mechanisms that may underlie their unique contributions to balance, movement, affect and cognition. This technique may also be used to explore differences in pathological susceptibility of these specific regions across various mouse disease models.

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression.&#160;

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...