Name: localization of the locus coeruleus in the mouse brain
Uploaded: 2019-03-07T14:00:00
Description: Watch this Scientific Journal Video about Localization of the Locus Coeruleus in the Mouse Brain at JoVE.com

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.

Studies of animals was approved by Johns Hopkins University Animal Care and use (ACUC) protocol number M017M385.
1. Brain Slicing
<ol>
	<li>To immobilize, anesthetize mice by the application of 3% isoflurane.
	<ol>
		<li>Soak a cotton ball with drops of isoflurane and place it in a 15 mL microcentrifuge tube. Place the animal&#8217;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.</li>
	</ol>
	</li>
	<li>Place ...

<ol>
	<li>Robertson, S. D., Plummer, N. W., de Marchena, J., Jensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origins+of+central+norepinephrine+neuron+diversity.">Developmental origins of central norepinephrine neuron diversity.</a> Nature neuroscience. 16, 1016-1023 (2013).</li><li>Kobayashi, R. M., Palkovits, M., Jacobowitz, D. M., Kopin, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+mapping+of+the+noradrenergic+projection+from+the+locus+coeruleus.+A+model+for+studies+of+brain+neuronal+pathways.">Biochemical mapping of the noradrenergic projection from the locus coeruleus. A model for studies of brain neuronal pathways.</a> Neurology. 25, 223-233 (1975).</li><li>Olson, L., Fuxe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+projections+from+the+locus+coeruleus+noradrealine+neurons:+the+cerebellar+innervation.">On the projections from the locus coeruleus noradrealine neurons: the cerebellar innervation.</a> Brain research. 28, 165-171 (1971).</li><li>Costa, A., Castro-Zaballa, S., Lagos, P., Chase, M. 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M., Tuppy, M., Moreira, T. S., Takakura, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+locus+coeruleus+catecholaminergic+neurons+in+the+chemosensory+control+of+breathing+in+a+Parkinson's+disease+model.">Role of the locus coeruleus catecholaminergic neurons in the chemosensory control of breathing in a Parkinson's disease model.</a> Experimental neurology. , 172-180 (2017).</li><li>Zarow, C., Lyness, S. A., Mortimer, J. A., Chui, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+loss+is+greater+in+the+locus+coeruleus+than+nucleus+basalis+and+substantia+nigra+in+Alzheimer+and+Parkinson+diseases.">Neuronal loss is greater in the locus coeruleus than nucleus basalis and substantia nigra in Alzheimer and Parkinson diseases.</a> Archives of neurology. 60, 337-341 (2003).</li><li>Chandley, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+deficits+in+pontine+locus+coeruleus+astrocytes+in+men+with+major+depressive+disorder.">Gene expression deficits in pontine locus coeruleus astrocytes in men with major depressive disorder.</a> Journal of psychiatry &amp; neuroscience : JPN. 38, 276-284 (2013).</li><li>Bernard, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+expression+of+glutamate+signaling,+growth+factor,+and+glia+genes+in+the+locus+coeruleus+of+patients+with+major+depression.">Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression.</a> Molecular psychiatry. 16, 634-646 (2011).</li><li>Gos, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+hydroxylase+immunoreactivity+in+the+locus+coeruleus+is+elevated+in+violent+suicidal+depressive+patients.">Tyrosine hydroxylase immunoreactivity in the locus coeruleus is elevated in violent suicidal depressive patients.</a> European archives of psychiatry and clinical neuroscience. 258, 513-520 (2008).</li><li>Bielau, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+evidence+for+impaired+nitric+oxide+signaling+of+the+locus+coeruleus+in+bipolar+disorder.">Immunohistochemical evidence for impaired nitric oxide signaling of the locus coeruleus in bipolar disorder.</a> Brain research. 1459, 91-99 (2012).</li><li>Wiste, A. K., Arango, V., Ellis, S. P., Mann, J. J., Underwood, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norepinephrine+and+serotonin+imbalance+in+the+locus+coeruleus+in+bipolar+disorder.">Norepinephrine and serotonin imbalance in the locus coeruleus in bipolar disorder.</a> Bipolar disorders. 10, 349-359 (2008).</li><li>Borodovitsyna, O., Flamini, M. D., Chandler, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Stress+Persistently+Alters+Locus+Coeruleus+Function+and+Anxiety-like+Behavior+in+Adolescent+Rats.">Acute Stress Persistently Alters Locus Coeruleus Function and Anxiety-like Behavior in Adolescent Rats.</a> Neuroscience. 373, 7-19 (2018).</li><li>Hirschberg, S., Li, Y., Randall, A., Kremer, E. J., Pickering, A. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nature. 13, 1526-1533 (2010).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosterone+administration+up-regulated+expression+of+norepinephrine+transporter+and+dopamine+beta-hydroxylase+in+rat+locus+coeruleus+and+its+terminal+regions.">Corticosterone administration up-regulated expression of norepinephrine transporter and dopamine beta-hydroxylase in rat locus coeruleus and its terminal regions.</a> Journal of neurochemistry. 128, 445-458 (2014).</li><li>Xiao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+regulates+rest-activity+cycles+through+the+locus+coeruleus-norepinephrine+system.">Copper regulates rest-activity cycles through the locus coeruleus-norepinephrine system.</a> Nature chemical biology. 14, 655-663 (2018).</li><li>Amaral, D. 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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 &#956;m 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...

An erratum was issued for: <a href="https://www.jove.com/video/58652/localization-of-the-locus-coeruleus-in-the-mouse-brain">Localization of the Locus Coeruleus in the Mouse Brain</a>.&#160; An author affiliation&#160;was updated.
The affiliation for Evan Maxey was updated from:
Department of Neuroscience, Johns Hopkins University
to:
X-ray science division, Advanced Photon Source, Argonne National Laboratory

An erratum was issued for: <a href="https://www.jove.com/video/58652/localization-of-the-locus-coeruleus-in-the-mouse-brain">Localization of the Locus Coeruleus in the Mouse Brain</a>.&#160; An author affiliation&#160;was updated.

Erratum: Localization of the Locus Coeruleus in the Mouse Brain

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 &#956;m, 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).

<table>
	<tbody>
		<tr>
			<td>Adult mouse brain slicer matrix</td>
			<td>Zivic Instruments</td>
			<td>BSMAS001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody, Alexa Fluor 488 (source - donkey)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Charged glass slides</td>
			<td>Genesee</td>
			<td>29-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microm GmbH</td>
			<td>HM 505E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat cutting blades</td>
			<td>Thermo Fisher Scientific</td>
			<td>MX35</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Mini, 9.5cm</td>
			<td>Antech Diagnostcs</td>
			<td>503241</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine &#946;-hydroxylase (DBH) antibody - inhouse production (source - rabbit)</td>
			<td>B. Eipper</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine &#946;-hydroxylase (DBH) antibody - commercially availabe (source - rabbit)</td>
			<td>Cell Signaling</td>
			<td>8586</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50ml</td>
			<td>USA Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Forane (isofluorane)</td>
			<td>Baxter</td>
			<td>NDC 1019-360-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Micro Adson</td>
			<td>Antech Diagnostcs</td>
			<td>501245</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardset mounting media</td>
			<td>EM sciences</td>
			<td>17984-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Pascal</td>
			<td>LSM 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well plates, 24 wells</td>
			<td>Thermo Fisher Scientific</td>
			<td>930186</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature compound (OCT)</td>
			<td>VWR/ tissue tech</td>
			<td>102094-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)/ formalin 10%</td>
			<td>Fisher Scientific</td>
			<td>SF98-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way disposable embedding molds</td>
			<td>Polysciences Inc.</td>
			<td>18646A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencil brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Tech</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Amazon</td>
			<td colspan="2">ASIN: B000CMFJZ2</td>
		</tr>
		<tr>
			<td>Spatulas</td>
			<td>Antech Diagnostcs</td>
			<td>14374</td>
			<td></td>
		</tr>
		<tr>
			<td>T pins</td>
			<td>Office Depot</td>
			<td>344615</td>
			<td></td>
		</tr>
		<tr>
			<td>The Mouse Brain in Stereotaxic Coordinates, Paxinos and Franklin, 3rd Edition</td>
			<td>Amazon</td>
			<td>ISBN: 978-0123694607</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100 (to prepare PBSD)</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosine hydroxylase (TH) antibody (source - rabbit)</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultralene thin film for XRF</td>
			<td>SPEX Sample Prep</td>
			<td>3525</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom.V16</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization of the locus coeruleus in the mouse brain

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-&#946;-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).

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. (<strong clas...

Watch this Scientific Journal Video about Localization of the Locus Coeruleus in the Mouse Brain at JoVE.com

Localization of the Locus Coeruleus in the Mouse Brain

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 ...

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.

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