Neuroscience
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Microdissection of Mouse Brain into Functionally and Anatomically Different Regions
Chapters
Summary February 15th, 2021
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.
Transcript
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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