Localization of the Locus Coeruleus in the Mouse Brain
Summary
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.
Abstract
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).
Introduction
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 neurological and neuropsychiatric disorders11, including Parkinson disease12,13,14, Alzheimer disease14, depression15,16,17, bipolar disorder18,19, and anxiety20,21,22,23,24. Given these roles, analysis of LC is crucial to studying its function and dysfunction.
Mice are widely used for studies of physiologic and pathophysiologic processes. Due to their small size, the mouse LC has an average diameter of ~300 μm, leading to difficulty locating the structure. During brain sectioning, the LC can easily be missed in either coronal or sagittal sections. Available studies describing identification of LC in animals do not offer a step-by-step protocol that a non-expert can follow1,25. Thus, to offer guidance for the localization of LC, we describe a protocol that we developed to locate this region in the mouse brain for several applications (Figure 1, Figure 2, Figure 3). The protocol applies carefully controlled brain sectioning and immunohistochemical detection of DBH26,27, or alternatively TH24, both enzymes highly enriched in the LC28. Once LC is located by immunohistochemistry, adjacent brain slices can be used for further studies, including morphological and metabolic analyses, as well as metal imaging studies via X-ray fluorescence microscopy (XFM)29. We describe XFM as an example in this protocol (Figure 3).
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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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