Dysfunction of locus coeruleus has been implicated in neurological and neuropsychiatric disorders, including Parkinson disease, Alzheimer disease, depression, bipolar disorder, and anxiety. Given these roles, analysis of locus coeruleus is crucial to studying its function and dysfunction. During mouse brain sectioning, the locus coeruleus can be easily missed in either coronal or sagittal sections due to its small size.
Thus, to offer guidance for the localization of locus coeruleus, we describe a protocol that we developed to locate this region in the mouse brain for several applications. Start by placing freshly isolated brain of an anesthetized and then perfused mouse in 4%PFA in a 50-milliliter tube. Incubate the brain for 24 hours at four degrees Celsius.
Use forceps to transfer the brain into a 50-milliliter conical tube filled with 25 milliliters of 30%sucrose solution. Incubate it at four degrees Celsius for 48 to 72 hours until the brain sinks to the bottom of the tube. To embed the brainstem section, place its cut surface on the bottom of an embedding mold, and add optimal cutting temperature compound to surround the brainstem.
Freeze the embedded brain in a minus 80 degree Celsius freezer for at least 12 hours until further use. Place the mold with the brain into the cryostat and incubate it for several hours to adjust its temperature to that of the cryostat. To expose the OCT block containing the brain, use a razor blade to cut the four edges of the mold all the way to the bottom, and then peel away the embedding mold from it.
Use razor blades to remove excess of OCT from the surface of the block, making sure not to touch the brain. Then mount the OCT block on the chuck of the cryostat, exposing the cut surface of the brain towards the front. To adjust the brain, orient the cut surface parallel to the razor blades of the cryostat.
Properly orienting the specimen is a crucial step in this protocol. Because we are using anatomical features of the dorsal surface of the brain to locate LC, such as boundary between cerebellum and inferior colliculus, it is important that the sections be aligned properly. This requires care in properly setting the brain into the mouse brain slicer matrix.
Trim the brain beginning at the medulla, cutting 100-micrometer sections rostrally. Once the cerebellum and brainstem start cutting as one continuous slice at the level of fourth ventricle, begin collecting slices at 50-micrometer thickness. Use forceps to collect each brain slice and place it in a well of a 24-well plate filled with PBS.
The locus coeruleus will be most noticeable in a slice where the cerebellum and inferior colliculus meet one another at about 5.52 millimeters posterior of bregma. On the first day, wash the brain slices in the 24-well plate three times for five minutes each in PBS. Then add 0.5%PBS with detergent and permeabilize them at four degrees Celsius for 24 hours.
On the following day, wash the slices three times for five minutes each with 0.5%PBSD. Then add the primary antibody at a dilution of one to 500 in 0.5%PBSD and incubate at four degrees Celsius for 18 hours. On the third day, wash the slices three times for 10 minutes each with 0.5%PBSD, then add the secondary antibody at a dilution of one to 1000 in 0.5%PBSD.
Wrap the plate with aluminum foil and incubate at four degrees Celsius for 16 hours. After incubation, wash the slices three times for five minutes each with 0.5%PBSD, and then for five minutes in PBS. Cover the sections using hard-set mounting medium without DAPI.
Cover with a glass cover and dry for 30 minutes at room temperature. Then use a confocal microscope with settings to detect signal from appropriate fluorescence wavelength to image brain slices. After adjusting the microscope to the focal plane of the brain slice, use 10 times magnification to take a single image.
Use the fourth ventricle located below the cerebellum and above pons and brainstem to locate a possible LC region in the brain slice. Focus on the lateral edges of the fourth ventricle and the LC will be located starting from the edges of the fourth ventricle and pointing toward the pons brainstem region. Intracellular distribution of dopamine beta-hydroxylase, a protein expressed in the LC, was assessed using this protocol in order to study LC morphology and neuronal density.
Another protein expressed in the LC, tyrosine hydroxylase, also showed a strong green fluorescent signal in brain slices containing the LC.Changes in metal homeostasis are often observed in neurologic disorders, including changes in the LC.In this example, when a brain slice cut through the LC was measured for phosphate, potassium, zinc, and copper, only copper showed a specific increase of the signal. We recommend cutting around 500 micron more tissue anterior and posterior to LC to avoid missing the nucleus, and cutting more sections than necessary, especially if first time performing this protocol. Careful study of the brain atlas images prior to staining is very helpful.
Once LC is located by immunohistochemistry, adjacent brain slices can be used for further studies, including morphological and metabolic analysis, as well as metal imaging studies via x-ray fluorescence microscopy. The brain sections generated using the described protocol can be used to quantify the levels of copper and other metals in the LC and compare them to the levels in regions outside of the LC, which is necessary for understanding of disease mechanism. Further possible applications of this protocol include the detection of abundance and intracellular distribution of dopamine beta-hydroxylase, tyrosine hydroxylase, and other proteins expressed in the LC individually, or in the co-staining assays, studies of LC morphology and neuronal density.
