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>

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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...

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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 ...

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42.4K 조회수.  The Geneva Foundation, Medical Readiness Systems Biology, Walter Reed Army Institute of Research. 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.

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

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.

총 RNA의 분리 및 다운 스트림 차세대 시퀀싱 및 유전자 발현 프로 파일링에 대한 개별 마우스 뇌 영역의 Microdissection 아닌 레이저 캡처 현미경 접근법

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

연구

신경과학

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

뇌의 작은, 해부학적인 몸의 구조 정의 분야의 DNA Methylation과 유전자 발현의 최적화 분석

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

동시 멕 / EEG와 해부학적인 몸의 구조 제약 최소 - 표준 견적을 사용하여 매핑 두피 역학 : 청각주의 예

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

성인 쥐의 뇌의 기존 및 비 인습적 인 지역에서 신경 줄기 세포 성장

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

마우스 뇌의 다양한 지역에서 텔로 머라 제 활동 : 비 방사성 텔로 머라 제는 증폭 프로토콜을 반복 (TRAP) 분석

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

설계, 치료, 세포 도금 및 기능적으로 간 연결 회로의 구성, 모듈 형 신경 네트워크의 배양 표면

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

마우스 해마에 올리고머 아밀로이드 - 베타의 정위 주입

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

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

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

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

청각 차별 학습 한 후 마우스 뇌 영역의 높은 해상도 양적 시냅틱 프로테옴 프로파일

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

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

신경 시스템 재구성, 변조 및 모델링을 위해 해부학 적으로 영감을받은 3 차원 미세 조직 공학 신경 회로망

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

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.