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Neuroscience

Localization of the Locus Coeruleus in the Mouse Brain

Published: March 7th, 2019

DOI:

10.3791/58652

1Department of Physiology, Johns Hopkins University, School of Medicine, 2Department of Neuroscience, Johns Hopkins University, 3Department of Molecular and Medical Genetics, OHSU, 4X-ray science division, Advanced Photon Source, Argonne National Laboratory

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-β-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus (LC) is an important region in the brainstem and a major site of norepinephrine (NE) production1. The LC sends projections throughout the brain2 to the cortex, the hippocampus and the cerebellum3 and regulates major physiological processes, including circadian rhythm4,5, attention and memory6, stress7, cognitive processes8, and emotion9,10. Dysfunction of LC has been implicated in neurologic....

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Studies of animals was approved by Johns Hopkins University Animal Care and use (ACUC) protocol number M017M385.

1. Brain Slicing

  1. To immobilize, anesthetize mice by the application of 3% isoflurane.
    1. Soak a cotton ball with drops of isoflurane and place it in a 15 mL microcentrifuge tube. Place the animal’s nose into the tube and allow it to inhale the isoflurane. Check for the depth of anesthesia by the lack of response to toe-pinch.
  2. Place .......

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Changes in metal homeostasis (such as Cu, Fe, Zn, and Mn) are often observed in neurologic disorders, including changes in the LC34,35. Thus, determining metal levels in the brain is necessary for understanding of disease mechanisms. The brain sections generated using the described protocol can be used to quantify the levels of Cu and other metals in the LC and compare them to the levels in regions outside of the LC. (

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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 (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. We recommend cutting ~500 μm more tissue anterior and posterior to LC to avoid missing the nucleus. The most common mistake is to cut too few sections that results in mi.......

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We thank Abigael Muchenditsi for the maintenance of the mouse colony. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number: DE-AC02-06CH11357. We thank Olga Antipova and Dr. Stefan Vogt for user support and assistance at the Advanced Photon Source. This work was funded by the National Institute of Health grant 2R01GM101502 to SL.

....

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Name Company Catalog Number Comments
Adult mouse brain slicer matrix Zivic Instruments BSMAS001-1
Anti-rabbit secondary antibody, Alexa Fluor 488 (source - donkey) Thermo Fisher Scientific A-21206
Charged glass slides Genesee 29-107
Confocal microscope Zeiss LSM 800
Cryostat Microm GmbH HM 505E
Cryostat cutting blades Thermo Fisher Scientific MX35
Scissors Mini, 9.5cm Antech Diagnostcs 503241
DAPI (4',6-diamidino-2-phenylindole) Sigma-Aldrich D9542-10MG
Dopamine β-hydroxylase (DBH) antibody - inhouse production (source - rabbit) B. Eipper -
Dopamine β-hydroxylase (DBH) antibody - commercially availabe (source - rabbit) Cell Signaling 8586
Falcon tubes, 50ml USA Scientific 339652
Forane (isofluorane) Baxter NDC 1019-360-60
Forceps Micro Adson Antech Diagnostcs 501245
Hardset mounting media EM sciences 17984-24
Microscope Pascal LSM 5
Multi-well plates, 24 wells Thermo Fisher Scientific 930186
Optimal cutting temperature compound (OCT) VWR/ tissue tech 102094-106
Paraformaldehyde (PFA)/ formalin 10% Fisher Scientific SF98-4
Peel-A-Way disposable embedding molds Polysciences Inc. 18646A
Pencil brush
Phosphate buffered saline (PBS) Life Tech 14190250
Razor blades Amazon ASIN: B000CMFJZ2
Spatulas Antech Diagnostcs 14374
T pins Office Depot 344615
The Mouse Brain in Stereotaxic Coordinates, Paxinos and Franklin, 3rd Edition Amazon ISBN: 978-0123694607
Triton-X 100 (to prepare PBSD) Sigma-Aldrich T8787
Tween 20 Sigma-Aldrich P7949-500ml
Tyrosine hydroxylase (TH) antibody (source - rabbit) EMD Millipore AB152
Ultralene thin film for XRF SPEX Sample Prep 3525
Wide-field fluorescent microscope Zeiss Axio Zoom.V16

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