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 p...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain Removal</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&#34; straight sharp/sharp</td>
		</tr>
		<tr>
			<td>Deaver scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6763</td>
			<td>5.5&#34; curved sharp/sharp</td>
		</tr>
		<tr>
			<td>Delicate operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6703</td>
			<td>4.75&#34; curved sharp/sharp</td>
		</tr>
		<tr>
			<td>Delicate operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6702</td>
			<td>4.75&#34; straight sharp/sharp</td>
		</tr>
		<tr>
			<td>Light operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6753</td>
			<td>5&#34; 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&#34;</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&#8221; 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&#34; 2X8mm</td>
		</tr>
		<tr>
			<td>Pituitary Dissection</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&#34;</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>Brain Dissection</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 &#38; 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&#8221; (10 cm) long</td>
		</tr>
		<tr>
			<td>Light operating scissors</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6753</td>
			<td>5&#34; 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&#34;</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&#8221; 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 ...

Studying distinct brain regions is a stepping stone towards comprehending the molecular and structural complexity of the brain. This protocol describes a systematic dissection of mouse brain to isolate distinct brain regions. In the perspective of brain anatomy, it is a top-down approach that can be easily reproduced.The advantages of this protocol are twofold. First, this approach provides an opportunity to understand molecular underpinnings of small, but critical brain regions. Second, this entire dissection process is sufficiently short to ensure preservation of molecular integrity, a critical prerequisite of back and molecular assays.This procedure involves practice and delicate handling. The timing is very crucial here to maintain the structural landmarks, and can be achieved with proper training and practice. Demonstrating the procedure of mouse brain removal will be Seid Muhie, a research chemist and bioinformatician from our lab.Further demonstrating the procedure of mouse brain dissection will be James Meyerhoff, a neuroscientist from our lab. To begin, clean and remove the skin for a good grip. Clamp the maxilla of the decapitated head with a hemostat and use a gauze to reflect the scalp rostrally.Insert the fine curved scissors into the foramen magnum to separate the adhering meninges. Then insert the scissors at the opening of the base of the skull where the spinal cord passes with the blade pointing vertically, but parallel to and pressing against the interior surface of the basal plate bone, rotating the blade 45 degrees to the left side and then to the right side. Remove the occipital bone in the intraparietal bone to expose the cerebellum.Advance the scissors rostrally along the mid sagittal suture up to the bregma, and cut it by lifting upward to avoid laceration of cerebral cortices. As the parietal bones are lifted, identify and cut the remaining meningeal attachments. Make two parallel cuts in the sagittal plane and remove the fragments of the frontal bone, avoiding lacerating the brain surface.While cutting meningeal attachments and cranial nerves, invert the skull to allow gravity to assist in the removal of the brain into a small cup pre-filled with saline solution. Keep auditory bulla intact for the pituitary anatomy to be identifiable. Make an extremely small parasaggital cut on both sides in the ridge of the membraneous tent that is holding pituitary in its place, and lift the posterior lobe of pituitary with ultra fine forceps.Make a sagittal cut between the lateral margins of the anterior lobe of pituitary in the nearest trigeminal nerve, then lift out the anterior lobe of pituitary. Direct the source of white light over the tissue. Place the brain on a pre-cooled stainless steel block and keep the block cold by surrounding it with ice in an ice-cold saline solution.Periodically moisten the tissue with ice-cold saline solution to preserve the structures. Position the brain such that the cerebral cortices are facing upwards. Using small curved forceps, remove the cerebellum.Using the dorsal approach, slip the closed blades of a small curved forceps beneath the corpus callosum, and gently spread it to retract the neocortex bilaterally to preserve the critically-salient midline landmarks. Eventual side up, structures such as optic chiasm, hypothalamus, olfactory bulbs, medulla oblongata, pituitary stock, and pontine bridge would be visible. Try separating the hemispheres.Flip the brain ventral site up and make a mid sagittal cut starting from the dorsum in between the olfactory bulbs in the cerebral hemispheres. Remove medulla, and then gently separate the two hemispheres. Next, make a coronal cut at the interior margin of the ponds to obtain the hind brain and separate the medulla at the posterior margin of the ponds.Flip the brain tissue for careful separation of the two cerebral hemispheres. Remove the olfactory bulb at this step. Make a coronal cut one millimeter anterior to the genu of the corpus callosum, and remove the accessory olfactory bulbs, followed by separating the medial and lateral prefrontal cortex.Partially cut horizontally or coronally 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 ventral striatum ventrally. Remove the small amount of the cortex from the lateral surface to remove the nucleus succumbence and olfactory tubercle. Separate the rostral portion of the corpus striatum from the overlying cortex via a curved scalpel cut just outside the external capsule.Identify the septal nuclei or septum and isolate them from this section. Make a curved cut to separate the hypothalamus at this step. This will be done using a partial coronal cut posterior to the mammillary bodies extending only as far laterally as the hypothalamic sulcus.Then free the hypothalamus cross-section with a parasagittal cut along the length of the hypothalamic sulcus. Evert the lateral ventricle to reveal additional intralimbic connections in the remaining limbic system components. On the inside part of the internal cortex, a fan-like structure is observed after opening the ventricle.Use the fan-like structure in the entorhinal cortex to define the margins for the purpose of dissection, then identify and remove the amygdala, entorhinal cortex, posterior cingulate cortex, and hippocampus. Confirm the identification of the thalamus by visualization of the stria medullaris on its dorsal surface extending in the midline rostral cortical plane. Separate the thalamus completely from the mid-brain by a coronal cut.This protocol can be used for immediate removal of the brain from the skull, followed by dissection. The dissected tissues can be preserved and processed later depending on the requirements of the study. Extracted RNA from multiple dissected tissues can be assayed for gene expression.The representative results were obtained using harvested brains that were stored at minus 80 degrees Celsius for several months before the RNA extraction. Distinct brain regions along with weight data were collected under the study aggressor exposed social stress model of PTSD. RNA concentrations and spectrophotometric readings are shown here.When following this protocol, ensure that the tissues are chilled at all times by keeping the brain on an iced steel block, and periodically dousing the tissue with iced saline. Once tissues are collected and immediately frozen, they can be used for assays like transcriptomics, metabolomics, and proteomics at later time.

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Anatomy of the Central and Peripheral Nervous System

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39.8K Views.  The Geneva Foundation, Medical Readiness Systems Biology, Walter Reed Army Institute of Research. Studying distinct brain regions is a stepping stone towards comprehending the molecular and structural complexity of the brain. This protocol describes a systematic dissection of mouse brain to isolate distinct brain regions. In the perspective of brain anatomy, it is a top-down approach that can be easily reproduced.The advantages of this protocol are twofold. First, this approach provides an opportunity to understand molecular underpinnings of small, but critical brain regions. Secon...