We thank Ms. Seshmalini Srinivasan, Mr. Stephen Butler and Ms. Pamela Spellman for experimental assistance and Ms. Dana Youssef for editing the manuscript. The funding support from USAMRDC is gratefully acknowledged. The Geneva Foundation contributed to this work and was supported by funds from the Military and Operational Medicine Research Area Directorate III via the US Army Research Office.
Disclaimer:
Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted under an approved animal use protocol in an AAALAC&#160;accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.

Animal handling and experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the US Army Center for Environmental Health Research now Walter Reed Army Institute of Research (WRAIR) and performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 
NOTE: The procedure will be performed on six to eight week old male mice of the C57BL/6j strain euthanized by cervical dislocation16. No perfusions are performed in our lab but this protocol could&#160;be modified whereby perfusions to clear blood from the vasculature could&#160;be performed. All supplies required for the dissection are listed in the Table of Materials. The dissection is subdivided into three components, including&#160;removal of the brain, the removal of the pituitary gland and the brain dissection. The intent of brain tissue collection is to process them for transcriptomic assays following RNA extractions. As soon as the brain region is dissected, we immediately transfer each of the brain regions into an already labeled freezer vial and then store the vial in liquid nitrogen or -80 &#176;C. 
NOTE: Thorough knowledge of neuroanatomy is essential to perform this procedure. Horizontal and longitudinal sections, as well as the usual transverse sections, should be studied and learned. Since brain tissue degrades very quickly, there is no time to consult an atlas while performing this procedure.
1. Mouse Brain Removal
<ol>
	<li>Clamp the maxilla of the decapitated head with a hemostat (Figure 2i) and use a gauze pad to reflect the scalp rostrally further creating a dry field on the dorsum of skull (Figure 2ii).</li>
	<li>Insert the fine curved scissors into the foramen magnum to separate the adhering meninges. Here, insert the scissors at the opening on the base of skull is the foramen magnum, where the spinal cord passes with the blade pointing vertically (12 o&#39;clock position), but parallel to and pressing against the interior surface of the basal plate bone (i.e., occipital squama) and rotating the blade forty-five&#176; to the left side and then to the right side.</li>
	<li>Continue to rotate the wrist to pry off the basal plate bone that will snip up the middle of the remaining bone continuing into the occipital bone and intraparietal bones prying the bones left and right until they are removed. In a similar fashion, remove the occipital bone and the intraparietal bone to expose the cerebellum. 
	NOTE: At this point, the hollow bony structure called the tympanic bullae, on the ventral posterior portion of the skull that encloses parts of the middle and inner ear, can be removed unless one is dissecting the posterior and anterior lobes of pituitaries (Figure 2iii).</li>
	<li>Remove the muscle attachments to the temporal ridge using a curved sharp scissor blade. Place one limb of the curved sharp scissors under the lambdoid suture penetrating into the junction of the transverse and sagittal sinuses (Figure 2iv).</li>
	<li>Advance the scissors rostrally along the midsagittal suture up to the bregma and cut it very gently while carefully lifting upward to avoid laceration of cerebral cortices. This is a critical step in the procedure (Figure 2v).</li>
	<li>At this step, parietal bones are lifted and rotated thus scraping the inner surface of the bone to identify and cut remaining meningeal attachments. Grasp the temporal bone prying outwardly, away from brain. Remove the frontal bone from each side using curved scissors or a rongeur into the orbit and cutting coronally at a right angle to the orbital ridge, but no further than the midline (Figure 2vii).</li>
	<li>Make two cuts parallel and about 4 mm apart in the sagittal plane (Figure 2viii). 
	NOTE: Do not cut both sides with one single stroke.</li>
	<li>Remove the fragments of the frontal bone avoiding lacerating the brain surface (Figure 2ix). At this point, use a fine scissors to cut the dura mater, which should be accessible between the olfactory bulbs.</li>
	<li>Gently invert the skull to allow gravity to assist in removal of the brain, while continuing to identify and cut remaining meningeal attachments and cranial nerves. The brain will be released by cutting the largest trigeminal nerve attached to the brain and is clearly visible extending from the base of the calvarium (Figure 2x). 
	&#8203;NOTE: Transfer the brain to a cold saline solution (ice cold RNase-free sodium citrate (0.9%) or physiological (0.9%) saline) for further dissection.</li>
</ol>
2. Dissection of the Anterior and Posterior Pituitaries
NOTE: The pituitary glands are covered by a very tough tent-like membrane, with a ridge that runs laterally between the left and right trigeminal nerves. These structures are extremely soft and delicate and, as such, it is recommended the posterior and anterior lobes of pituitary glands be dissected separately in stages, in situ, directly from the skull. Immediately after dissection, transfer the respective pituitary gland to a pre-labeled vial and store the vial in liquid nitrogen preferably otherwise -80 &#176;C. The pituitary gland rests exactly over the junction of the occipital and basisphenoid bones; if they flex, the pituitary architecture is disrupted.
<ol>
	<li>Keep the auditory bullae intact to make sure that the pituitary anatomy is intact and easily identifiable (Figure 2ix).</li>
	<li>Dissect the posterior lobe of pituitary followed by the anterior lobe of pituitary, from the remainder of the skull.</li>
	<li>Make an extremely small parasagittal cut on both sides in the ridge of the membranous tent and lift the posterior lobe of pituitary with ultra-fine forceps, taking care not to disrupt the anterior pituitary tissue (Figure 2x).</li>
	<li>Make a sagittal cut between the lateral margins of the anterior lobe of pituitary and the nearest trigeminal nerve and lift out the anterior lobe of pituitary thereafter (Figure 2x).</li>
</ol>
3. Mouse Brain Dissection
NOTE: Immediately after brain and pituitary removal, further dissection is performed on a pre-chilled stainless-steel block (Figure 3). Post dissection, transfer the brain regions to pre-labeled vials and transfer the vials preferably to liquid nitrogen otherwise -80 &#176;C. Structures produced by the top-down method (in chronological order) potentially include the following: cerebellum (CB), brain stem/hind brain (pons and medulla oblongata) (HB), olfactory bulbs (OB) as accessory olfactory bulbs, medial prefrontal cortex (MPFC), lateral prefrontal cortex (FCX), anterior and posterior corpus striatum (ST), ventral striatum (VS) comprised of the nucleus accumbens (NAC) and olfactory tubercle (OT), septum (SE), preoptic area, piriform cortex (PFM), hypothalamus (HY), amygdala (AY), hippocampus (HC), posterior cingulate cortex (CNG), entorhinal cortex (ERC), midbrain (MB) with thalamus and rest of the cerebral cortex (ROC) (Table 1). Specific regions will be discussed in order of isolation, working with a single hemisphere.
<ol>
	<li>Take great care in removing the brain from the calvarium as the landmarks may be destroyed in case there are any lacerations. At this step, perform all dissections using face protection, specifically, a 7x jewelers visor, and illumination will be provided by surgical lamps positioned over each of the dissector&#39;s shoulders. Much of the dissection will be accomplished using blunt mode small curved forceps (i.e., Graefe forceps).</li>
	<li>Place the brain on a stainless-steel block (Figure 4i and Figure 4ii). Keep the block cold by surrounding it with ice and an ice-cold saline solution. Periodically moist the tissue with ice-cold saline solution to preserve the structures.</li>
	<li>Position the brain such that the cerebral cortices are facing upwards. Using small curved forceps, gently reflect the CB by exposing the superior, middle and inferior cerebellar peduncles and remove the CB (Figure 4iii and Figure 4iv).</li>
	<li>Make a midsagittal cut starting from the dorsum and between the OB and the cerebral hemispheres (Figure 4v) and make sure not to extend farther than the anterior commissure. At this point, the vermis can be easily separated from the lateral portions and HB will be obtained by a coronal cut at the anterior margin of the pons.</li>
	<li>Separate the medulla by a coronal cut at the posterior margin of the pons. 
	NOTE: At this point, the diencephalon, the posterior part of the forebrain containing the epithalamus, thalamus, hypothalamus, and ventral thalamus and the third ventricle, will be turned ventral side up, and a midsagittal cut will be made from the optic chiasm rostrally. The dissection of cerebral hemispheres followed by removal of OB from one of the hemisphere will be at this step (Figure 4v and Figure 4vi).</li>
	<li>Separate the cerebral hemispheres (Figure 4v) before dissecting the diencephalon further (Figure 4v). The dorsal approach will be employed by careful blunt dissection to preserve the critically salient midline landmarks. Visualize every interhemispheric connection before severing them as this will minimize the possibility of deviating from the midline. 
	NOTE: At this step one of the cerebral hemisphere (Hemibrain) can be preserved and the second hemisphere can be used for further dissection</li>
	<li>Slip the closed blades of a small, curved forceps, beneath the corpus callosum and gently spread to retract the neocortex bilaterally. The corpus callosum is a broad band of nerve fibers that joins the two hemispheres which will be bluntly dissected by pinching with the forceps, without disturbing the midline structures lying beneath. 
	NOTE: Mesial faces of the hemispheres will have multiple landmarks visible such as myelinated structures like the genu of the corpus callosum, the fornices, and the anterior commissure. Also, though a few millimeters lateral to the midline, the mammillothalamic tract and the fasciculus retroflexus may also be visible.</li>
	<li>Bisect the multiple structures that cross the midline. This will include the corpus callosum, anterior commissure, ventral fornix commissure, posterior commissure, dorsal fornix commissure, supra mammillary decussation, superior colliculus commissure, ventral fornix commissure, and periventricular thalamic fibers.</li>
	<li>Take special care at this step in or near the midline that might be partially compromised by the dorsal approach method. All follow-up structures are at risk if not performed carefully.</li>
	<li>Remove the&#160;OB (wedge shaped and lighter color) and accessory olfactory bulbs followed by collection of the MPFC (cingulate cortex area 1, prelimbic, infralimbic, medial orbital, and secondary motor cortices [M2]) and FCX by a coronal cut that is made 1mm anterior to the genu of the corpus callosum (Figure 4vii). Divide the resulting section by a parasagittal cut 1/3 of the distance from the medial to the lateral surface, yielding MPFC medially and remainder of FCX laterally. The cingulate cortex is the mouse analog of MPFC. 
	NOTE: This tissue slice will also contain a small amount of the M2 (secondary motor cortex) 17 and is unavoidable.</li>
	<li>Make a coronal cut at the level of the anterior commissure leading to visibility of pars anterior limb of the anterior commissure in the cross-section on the rostral face of the resulting coronal section whereas the transverse portion will be revealed in the caudal face of the section This is a confirmatory landmark for NAC18. The VS is composed of the NAC and OT. 
	NOTE: There is an anterior limb in addition to the transverse segment in the anterior commissure and it is called anterior commissure, pars anterior.</li>
	<li>Partially cut horizontally through the transverse portion of the anterior commissure from the midline beneath the anterior horn of the lateral ventricle, to free the septal nucleus dorsally and the VS ventrally. Remove the small amount of the cortex from the lateral surface where NAC and OT will be removed.</li>
	<li>Separate the rostral portion of the ST from the overlying cortex via curved scalpel cut just outside the external capsule taking care not to include striatal tissue in the cortical sample. At this point, the septal nuclei /SE will be visible and can be easily taken from this slice (Figure 4viii). 
	NOTE: The anterior and posterior limits of the PFM are defined by imaginary coronal planes that are in line with the anterior commissure and the mammillary bodies respectively. The medial limit is defined by the external capsule18.</li>
	<li>On the lateral surface of the remaining hemi-section of the diencephalon, make a partial horizontal cut along the rhinal sulcus extending caudally, but only as far as the imaginary coronal plane level with the mammillary bodies. Make a partial coronal cut, extending medially 1 mm from the lateral cortical surface in the plane of the mammillary bodies of the HY. Here, para-sagittal incision in the plane of the claustrum will free the PFM (Figure 4ix and Figure 4x). 
	NOTE: On the medial surface of the remaining hemi-section of the diencephalon, numerous anatomical features will be visible including the mammillary bodies, the fasciculus retroflexus, and the stria medullaris. A semicircular pattern (dorsal concave side) will be visible, which denotes the margin between the thalamus and the HY. The HY will be easily identified on the midsagittal section view per the optic chiasm anteroventrally and anterior commissure anterodorsally and the mammillary bodies along with the fasciculus retroflexus posteriorly. The latter landmarks have been well displayed in the atlas17 as well as in albino mouse forebrain context19.</li>
	<li>Make a partial coronal cut posterior to the mammillary bodies, extending only as far laterally as the hypothalamic sulcus. At this point a parasagittal cut along the length of the hypothalamic sulcus now frees the HY .</li>
	<li>Entail the eversion of the lateral ventricle to reveal additional intra-limbic connections and the remaining limbic system components. Viewing the medial face of the remaining hemi-section of the diencephalon, curved forceps will be best option as they allow the technician to manipulate around other delicate sections of the surrounding area used to sever the fornix and is inserted into the lateral ventricle and gently expanded, using blunt dissection to open the ventricle and rotate the HC 90&#176; from the vertical to the horizontal plane. It may be necessary to use the #11 blade to section the choroidal artery/choroid plexus as the pointed tip can help with precise cuts.</li>
	<li>Rotate the CNG 90&#176; (but in the opposite direction from the HC) to be in the horizontal plane. Using forceps, gently rotate the HC another 180&#176; outward and laterally, which will make the inner surface of the ERC visible and facing upwards.</li>
	<li>A fan-like radiation of myelinated fibers arising from the ERC will be seen converging to form the angular bundle, including the perforant path and its attachment to the HC. Rotate the thalamus and MB 180&#176; dorsally to ventrally reveal the AY.</li>
	<li>If required, lift the optic tract to reveal the attachment of the stria terminalis as it will contain bands of fibers running along the lateral margin of the ventricular surface of the thalamus to the AY and will make the outlines of the AY clear. 
	NOTE: The HC and fornix will be clearly visible in their entirety, nested in the lateral ventricle and, can be easily lifted out. The lateral ventricle will be lined with pia mater and fan-like origins of the perforant path through the very transparent subpial aspect of the ERC will be visible20.</li>
	<li>The outer surface of medial ERC will have a visible prominent layer of large pyramidal cells. In addition, a dense convergence of Golgi-staining fibers will be a salient feature in the medial ERC. In this dissection procedure, after everting the lateral ventricle, these fibers will be easily visible on the medial surface through the pia mater, which lines the inner surfaces of the ventricles, forming a very prominent fan-like structure.</li>
	<li>Notice that the fibers are gathering subpially, descending and leaving the ventral ERC, extending posteriorly and then ascending vertically to perforate the HC. This fan-like structure in the ERC will be used to define the margins for the purpose of dissection. Identify and remove in the order of AY, ERC, CNG and HC structures. 
	NOTE: Defining a good landmark for AY dissection will be a key for the next step and the junction on posterior margin where stria terminalis meets the AY will be a good point.</li>
	<li>At this point, the remaining structures include the thalamus and the MB. Confirm the identification of the thalamus by visualization of the stria medullaris on its dorsal surface extending in the midline rostrocaudal plane. Separate the thalamus completely from the midbrain by a coronal cut caudal to the habenula and rostral to the superior colliculi. The ROC is saved at this step.</li>
	<li>At this point complete collection of all brain regions and, immediately (as each is obtained) store the samples flash frozen till further processing.</li>
</ol>

<ol>
	<li>Zeisel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Architecture+of+the+Mouse+Nervous+System.">Molecular Architecture of the Mouse Nervous System.</a> Cell. 174 (4), 999-1014 (2018).</li><li>Ackerman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Major Structures and Functions of the Brain. 2, (1992).</li><li>P, T. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2019).</li><li>Paramvir, T. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2019).</li><li>Javed, K., Reddy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy, Cerebral Cortex. , (2020).</li><li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+neocortex:+a+perspective+from+developmental+biology.">Evolution of the neocortex: a perspective from developmental biology.</a> Nature Reviews Neuroscience. 10 (10), 724-735 (2009).</li><li>Fern&#225;ndez, V., Llinares-Benadero, C., Borrell, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+cortex+expansion+and+folding:+what+have+we+learned.">Cerebral cortex expansion and folding: what have we learned.</a> The EMBO Journal. 35 (10), 1021-1044 (2016).</li><li>Pessoa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+brain+networks+and+brain+organization.">Understanding brain networks and brain organization.</a> Physics of Life Reviews. 11 (3), 400-435 (2014).</li><li>Mu, Q., Chen, Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+Brain+Complexity+Using+Single-cell+Sequencing.">Deciphering Brain Complexity Using Single-cell Sequencing.</a> Genomics, Proteomics &amp; Bioinformatics. 17 (4), 344-366 (2019).</li><li>Darmanis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+human+brain+transcriptome+diversity+at+the+single+cell+level.">A survey of human brain transcriptome diversity at the single cell level.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (23), 7285-7290 (2015).</li><li>Hodge, R. D., 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=Conserved+cell+types+with+divergent+features+in+human+versus+mouse+cortex.">Conserved cell types with divergent features in human versus mouse cortex.</a> Nature. 573 (7772), 61-68 (2019).</li><li>Winrow, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 Research. 1271, 1-17 (2009).</li><li>Atkins, N., Miller, C. M., Owens, J. R., Turek, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Journal of Visualized Experiments. (57), e3125 (2011).</li><li>Wager-Miller, J., Murphy Green, M., Shafique, H., Mackie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+Frozen+Rodent+Brain+Regions+for+Downstream+Analyses.">Collection of Frozen Rodent Brain Regions for Downstream Analyses.</a> Journal of Visualized Experiments. (158), e60474 (2020).</li><li>Muhie, S., 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=Brain+transcriptome+profiles+in+mouse+model+simulating+features+of+post-traumatic+stress+disorder.">Brain transcriptome profiles in mouse model simulating features of post-traumatic stress disorder.</a> Molecular Brain. 8, 14 (2015).</li><li>Hammamieh, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+repeated+exposures+to+conspecific+trained+aggressors+simulates+features+of+post-traumatic+stress+disorder.">Murine model of repeated exposures to conspecific trained aggressors simulates features of post-traumatic stress disorder.</a> Behavioural Brain Research. 235 (1), 55-66 (2012).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. Compact 3rd edn. , (2008).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Coronal Plates and Diagrams. , (2019).</li><li>Slotnick, B. M., Leonard, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+atlas+of+the+albino+mouse+forebrain.+Rockville,+MD,+Alcohol,+Drug+Abuse+and+Mental+Health+Administration,+1975.">Stereotaxic atlas of the albino mouse forebrain. Rockville, MD, Alcohol, Drug Abuse and Mental Health Administration, 1975.</a> Annals of Neurology. 10 (4), 403-403 (1981).</li><li>Cajal, S. R., Swanson, N., Swanson, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histologie Du Syst&#232;me Nerveux de L'homme Et Des Vert&#233;br&#233;s. Anglais. , (1995).</li><li>Spijker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Rodent+Brain+Regions.">Dissection of Rodent Brain Regions.</a> Neuromethods. 57, 13-26 (2011).</li><li>Wager-Miller, J., Murphy Green, M., Shafique, H., Mackie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+Frozen+Rodent+Brain+Regions+for+Downstream+Analyses.">Collection of Frozen Rodent Brain Regions for Downstream Analyses.</a> Journal of Visualized Experiments. (158), e60474 (2020).</li><li>Sultan, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Different+Areas+from+Mouse+Hippocampus.">Dissection of Different Areas from Mouse Hippocampus.</a> Bio Protocols. 3 (21), (2013).</li><li>Chakraborty, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+and+stress+history+interplay+in+emergence+of+PTSD-like+features.">Gene and stress history interplay in emergence of PTSD-like features.</a> Behavioural Brain Research. 292, 266-277 (2015).</li><li>Chiu, K., Lau, W. M., Lau, H. T., So, K. -. F., Chang, R. C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-dissection+of+rat+brain+for+RNA+or+protein+extraction+from+specific+brain+region.">Micro-dissection of rat brain for RNA or protein extraction from specific brain region.</a> Journal of Visualized Experiments. (7), (2007).</li><li>Rajmohan, V., Mohandas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+limbic+system.">The limbic system.</a> Indian Journal of Psychiatry. 49 (2), 132-139 (2007).</li></ol>

The authors have nothing to disclose.

The mammalian brain is a complex organ composed of an array of morphologically distinct and functionally unique cells with diverse molecular signatures and multiple regions that perform specialized and discrete functions. The dissection procedure reported here can have multiple goals depending on the requirements of the lab. In our lab we assessed transcription in multiple brain regions collected from mice exposed to PTSD like stress16 . We would like to study further the impact of strain genetic ...

The brain, along with the spinal cord and retina, comprise&#160;the central nervous system that executes complex behaviors, controlled by specialized, precisely positioned, and interacting cell types throughout the entire body1. The brain is a complex organ with billions of interconnected neurons and glia with precise circuitry performing numerous functions. It is a bilateral structure with two distinct lobes and diverse cellular components2. The spinal cord connects the brain to the outside world and is protected by bone, meninges, and cerebrospinal fluid and routes messages to and from the brain2,3,4. The surface of the brain, the cerebral cortex, is uneven and has distinct folds, called gyri, and grooves, called sulci, that separate the brain into functional centers5. The cortex is smooth in mammals with a small brain6,7. It is important to characterize and study the architecture of the human brain in order to understand the disorders related to the different brain regions, as well as its functional circuits. Neuroscience research has expanded in recent years and a variety of experimental methods are being used to study the structure and function of the brain. Developments in the fields of molecular and systems-level biology have ushered in a new era of exploring the complex relationship between brain structures and the functioning of molecules. Additionally, molecular biology, genetics, and epigenetics are rapidly expanding, enabling us to advance our knowledge of the underlying mechanisms involved in how systems function. These analyses can be carried out on a much more localized basis, to help target the investigation and development of more effective therapies.
The mammalian brain is structurally defined into clearly identifiable discrete regions; however, the functional and molecular complexities of these discrete structures are not yet clearly understood. The multi-dimensional and multi-layered nature of the brain tissue makes this landscape difficult to study at the functional level. In addition, the fact that multiple functions are performed by the same structure and vice versa further complicates the understanding of the brain8. It is vital that the experimental approach executed for the structural and functional characterization of brain regions uses precise research methodologies to achieve consistency in sampling for correlating neuroanatomical architecture with function. The complexity of brain has been recently explained using single cell sequencing9,10 such as the temporal gyrus of the human brain which is composed of 75 distinct cell types11. By comparing this data to those from an analogous region of the mouse brain, the study not only reveals similarities in their architecture and cell types but also presents the differences. To unravel the complex mechanisms, it is therefore important to study diverse regions of the brain with full precision. Conserved structures and function between a human and mouse brain enable the use of a mouse as a preliminary surrogate for elucidating human brain function and behavioral outcomes.
With the advancement of systems biology approaches, obtaining information from discrete brain regions in rodents has become a key procedure in neuroscience research.While some protocols such as laser&#160;capture&#160;microdissection12 can be expensive, mechanical protocols are inexpensive and performed using commonly available tools13,14. We have used multiple brain regions for transcriptomic assays15 and have developed a hands-on and rapid procedure to dissect mouse brain regions of interest in a step-by-step manner in a short time. Once dissected, these samples can be stored immediately in cold conditions to preserve the nucleic acids and proteins of these tissues. Our approach can be performed faster leading to high efficiency and permitting less chances for tissue deterioration. This ultimately, increases the chances of generating high quality, reproducible experiments using brain tissues.

<table><tbody><tr><td>&lt;strong&gt;Brain Removal&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Deaver scissors</td><td>Roboz Surgical Store</td><td>RS-6762</td><td>5.5&quot; straight sharp/sharp</td></tr><tr><td>Deaver scissors</td><td>Roboz Surgical Store</td><td>RS-6763</td><td>5.5&quot; curved sharp/sharp</td></tr><tr><td>Delicate operating scissors</td><td>Roboz Surgical Store</td><td>RS-6703</td><td>4.75&quot; curved sharp/sharp</td></tr><tr><td>Delicate operating scissors</td><td>Roboz Surgical Store</td><td>RS-6702</td><td>4.75&quot; straight sharp/sharp</td></tr><tr><td>Light operating scissors</td><td>Roboz Surgical Store</td><td>RS-6753</td><td>5&quot; curved Sharp/Sharp</td></tr><tr><td>Micro spatula, radius and tapered flat ends</td><td></td><td></td><td>stainless steel mirror finish</td></tr><tr><td>Operating scissors 6.5&quot;</td><td>Roboz Surgical Store</td><td>RS-6846</td><td>curved sharp/sharp</td></tr><tr><td>Tissue forceps</td><td>Roboz Surgical Store</td><td>RS-8160</td><td>4.5&amp;rdquo; 1X2 teeth 2mm tip width</td></tr><tr><td>Rongeur (optional)</td><td>Roboz Surgical Store</td><td>RS-8321 many styles to choose</td><td>Lempert Rongeur 6.5&quot; 2X8mm</td></tr><tr><td>&lt;strong&gt;Pituitary Dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Scalpel handle</td><td>Roboz Surgical Store</td><td>RS-9843</td><td>Scalpel Handle #3 Solid 4&quot;</td></tr><tr><td>and blades</td><td>Roboz Surgical Store</td><td>RS-9801-11</td><td>Sterile Scalpel Blades:#11 Box 100 40mm</td></tr><tr><td>Super fine forceps Inox</td><td>Roboz Surgical Store</td><td>RS-4955</td><td>tip size 0.025 X 0.005 mm</td></tr><tr><td>&lt;strong&gt;Brain Dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A magnification visor</td><td>Penn Tool Col</td><td>40-178-6</td><td>2.2x Outer and 3.3x Inner Lens Magnification, Rectangular Magnifier</td></tr><tr><td>Dissection cold plate</td><td>Cellpath.com</td><td>JRI-0100-00A</td><td>Iceberg cold plate &amp;amp; base</td></tr><tr><td>Graefe forceps, full curve extra delicate</td><td>Roboz Surgical Store</td><td>RS-5138</td><td>0.5 mm Tip 4&amp;rdquo; (10 cm) long</td></tr><tr><td>Light operating scissors</td><td>Roboz Surgical Store</td><td>RS-6753</td><td>5&quot; curved sharp/sharp</td></tr><tr><td>Scalpel handle</td><td>Roboz Surgical Store</td><td>RS-9843 (repeated above)</td><td>Scalpel Handle #3 Solid 4&quot;</td></tr><tr><td>and blades (especially #11)</td><td>Roboz Surgical Store</td><td>RS-9801-11 (repeated above)</td><td>Sterile Scalpel Blades:#11 Box 100 40mm</td></tr><tr><td>Spatula</td><td>Amazon</td><td>MS-SQRD9-4</td><td>Double Ended Spatula Square AND Round End</td></tr><tr><td>Tissue forceps</td><td>Roboz Surgical Store</td><td>RS-8160 (repeated above)</td><td>4.5&amp;rdquo; 1X2 teeth</td></tr></tbody></table>

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.

Our understanding of the complex brain structure and function is rapidly evolving and improving. The brain contains multiple distinct regions and building a molecular map can help us better understand how the brain works. In this method paper, we have discussed the dissection of the mouse brain into multiple distinct regions (Table 1). In this protocol, the structures are identified based on the critical landmarks and is achieved by keeping the tissue moist with saline solution by retaining its sturdines...

Watch this Scientific Journal Video about Microdissection of Mouse Brain into Functionally and Anatomically Different Regions at JoVE.com

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

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 ...

microdissection-mouse-brain-into-functionally-anatomically-different

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

Anatomy of the Central and Peripheral Nervous System

SCIENTISTS IN ACTION

44.7K Views.  The Geneva Foundation, Medical Readiness Systems Biology, Walter Reed Army Institute of Research. Het bestuderen van verschillende hersengebieden is een opstap naar het begrijpen van de moleculaire en structurele complexiteit van de hersenen. Dit protocol beschrijft een systematische dissectie van muizenhersenen om verschillende hersengebieden te isoleren. In het perspectief van de hersenanatomie is het een top-down benadering die gemakkelijk kan worden gereproduceerd.De voordelen van dit protocol zijn tweeledig. Ten eerste biedt deze benadering een kans om de moleculaire onderbouw...

Video: Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

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 ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

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.

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 ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

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 ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay

Source: Sumitomo, A., et al. <a href="https://app.jove.com/v/65063">BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain.</a> J. Vis. Exp. (2023)
This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leaving internal proteins unaltered. The reaction is quenched with glycine before processing the samples for further analysis.

Source: Sumitomo, A., et al. BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

Samenvatting

Meer video's verkennen