Name: Localization of the Locus Coeruleus in the Mouse Brain (Video) | JoVE Journal
Uploaded: 2019-03-07T14:00:00.000+00:00
Duration: 7 min 44 s
Description: Learn a precise protocol for localizing the locus coeruleus in mouse brain sections using immunohistochemistry and anatomical landmarks. This method enables analysis of protein expression and metal homeostasis in neurological research.

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5 minutes

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 the animal on its back and immobilize it by pinning its extremities down with a T pin while having access to its abdomen.</li>
	<li>Cut the animal with surgical scissors by making a snip from the abdominal skin and cut through the skin in the thorax region. Pin the skin down using T pins. Then break the peritoneal membrane up to the thorax. Expose the heart by cracking the thoracic cavity and cutting the diaphragm.</li>
	<li>Cut the right atrium to allow the blood to flow out of the animal. Insert a 10 mL syringe with a 25-gauge needle into the left ventricle and perfuse with 10 mL of phosphate-buffered saline (PBS). 
	NOTE: This allows the solution to flow through systemic circulation and exit through the right atrium.</li>
	<li>Remove the 10 mL syringe and insert a 25-gauge needle attached to the 60 mL syringe. Perfuse through the left ventricle with 50 mL of ice cold 4% paraformaldehyde (PFA).
	<ol>
		<li>Prepare ice cold 4% PFA solution by diluting 10% PFA solution in H2O and chilling the final 4% PFA solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Isolate the head of the mouse and remove the brain from the skull.
	<ol>
		<li>Cut the skin from the neck, and then cut towards the eyes to expose the skull. Crack the skull from the neck to the nose, and then from one eyeball to the other. Peel the skull out and excise the whole brain.</li>
	</ol>
	</li>
	<li>Incubate the brain in 4% PFA for 24 h at 4 &#176;C.</li>
	<li>Transfer the brain with forceps into a 50 mL conical tube filled with 25 mL of 30% sucrose solution. Keep it at 4 &#176;C for 48-72 h until the brain sinks to the bottom of tube.</li>
	<li>Cut the brain with an adult mouse brain slicer matrix coronal through the midbrain (~3 mm posterior of bregma). Keep the brain section containing the brainstem. 
	NOTE: This will result in two brain sections &#8211; one containing most of cortex (anterior of the cut) and one containing the brainstem/cerebellum (posterior of the cut). Use the brainstem section for the following steps.</li>
	<li>Embed the brainstem section with the cut surface placed on the bottom of an embedding mold, surrounded by optimal cutting temperature compound (OCT); move the embedded brain to a -80 &#176;C freezer and freeze for at least 12 h &#8211; until further use.</li>
	<li>Cutting at the cryostat: place the embedding mold containing the brain in OCT into the cryostat; incubate it in the cryostat for several hours to adjust the temperature of the brain block to that of the cryostat.</li>
	<li>Peel away the embedding mold to expose the OCT block containing the brain.</li>
	<li>Use razor blades to remove excess of OCT from the surface of the block without touching the brain.</li>
	<li>Mount the OCT block on the chuck of the cryostat, exposing the cut surface of the brain toward the front.</li>
	<li>Adjust the cut surface of the brain so that it is oriented in parallel to the razorblades of the cryostat.</li>
	<li>Trim the brain beginning at the medulla, cutting 100 &#181;m sections rostrally.</li>
	<li>Trim rostrally until the cerebellum and brainstem cut as one continuous slice. Begin collecting slices at 50 &#181;m thickness. 
	NOTE: As one trims rostrally from the medulla, the brainstem and cerebellum will cut as two separate sections. In rostral sections, the brainstem and cerebellum will eventually merge at the level of the 4th ventricle. Once the lateral edges of the 4th ventricle are well-formed, then the cerebellum and brainstem will come out as one continuous slice.
	<ol>
		<li>Collect OCT-surrounded brain slice with forceps and place it in a well of a 24-well plate filled with PBS (Figure 2a). The LC will be most prominent when the cerebellum and inferior colliculus meet one another at ~-5.52 mm posterior of bregma (Figure 1b). 
		NOTE: The most anterior part of LC will disappear once the cerebellum has been fully sectioned and no longer surrounds the inferior colliculus at ~-5.34 mm posterior of bregma (Figure 1c).</li>
	</ol>
	</li>
</ol>
2. Immunohistochemistry for Dopamine &#946;-Hydroxylase or Tyrosine Hydroxylase (Figure 2)
<ol>
	<li>Day 1
	<ol>
		<li>Wash the selected slices three times for 5 min in PBS.</li>
		<li>Permeabilize for 24 h in 0.5% phosphate buffered saline with detergent (PBSD) at 4 &#176;C.
		<ol>
			<li>Dilute 125 &#181;L of detergent in 25 mL of PBS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Wash slices three times for 5 min in 0.5% PBSD.</li>
		<li>Add the primary antibody, anti-DBH or anti-TH for 18 h at a dilution of 1:500 in 0.5% PBSD at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Wash slices three times for 10 min in 0.5% PBSD.</li>
		<li>Add the desired secondary antibody (488 donkey anti-rabbit for green fluorescence) at a dilution of 1:1000 in 0.5% PBSD for 16 h.</li>
		<li>Wrap the 24 well plate in aluminum and place at 4 &#176;C.</li>
		<li>Wash slices three times for 5 min in 0.5% PBSD.</li>
		<li>Wash for 5 min in PBS.</li>
		<li>Transfer slices with a pencil brush into a water container.</li>
		<li>Mount slices floating in water on charged slides.</li>
		<li>Coverslip sections with hard-set mounting media (without DAPI).</li>
		<li>Dry the mounted brain sections for 30 min at room temperature.</li>
		<li>Image brain slices at a confocal or fluorescent microscope with settings to detect signal from appropriate secondary antibody fluorophore wavelength.</li>
		<li>Adjust the microscope to the focal plane of the brain slice and take a single image at 10X magnification.</li>
		<li>To locate a possible LC region in the brain slice, use the 4th ventricle for orientation; the cerebellum is located above the ventricle, pons and brainstem are found below30.</li>
		<li>To locate the LC, focus on the lateral edges of the 4th ventricle; the LC originates from the edges of the 4th ventricle and points toward the pons/ brainstem region (Figure 2b, 2c).</li>
		<li>Following imaging and localization of the LC in certain brain slices, store slides containing brain slices at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3.&#160;Detection of the LC in Brain Slices
<ol>
	<li>To locate the brain section containing the LC, slice the mouse brain as described above and collect sections in PBS filled 24 well dishes, as shown in Figure 2a.
	<ol>
		<li>Place one brain section per well to allow for the proper localization of the LC.</li>
	</ol>
	</li>
	<li>Collect a total of 48 brain slices that will be immunostained per brain. 
	NOTE: All the wells of the two dishes shown in Figure 2a contain brain slices from one animal.</li>
	<li>Immunostain every third to fifth slice for DBH, or TH. Preferably, perform immunohistochemistry of those brain slices that are adjacent to those that are assayed further (via X-ray fluorescence microscopy, XFM). 
	NOTE: This procedure allows for exact localization of the LC in the final assay slices (Figure 2a; labeled with numbers between the wells).</li>
	<li>Adjust the number of brain slices that are immunostained depending on the application, e.g., whether they require exact location of the center and edge of the LC, or just a rough approximation.</li>
	<li>Following immunohistochemistry, detect brain slices containing LC via a characteristic pattern of expression at both sides of the 4th ventricle (Figure 2b, 2c). Magnification of the DBH signal in the LC of consecutive brain slices is shown in Figure 2d, 2e; the magnified image of the LC that is stained for TH is shown in Figure 2f.</li>
	<li>Use the sections adjacent to those containing LC for further studies - in this case for XFM to quantify metal levels (Figure 3).</li>
	<li>To perform XFM, collect 10-30 &#181;m (depending on the setup) thin coronal brain slices on thin polymeric film with which they can be mounted on sample holders and imaged at the synchrotron.</li>
	<li>Store brain slices which are prepared on thin polymeric film for XFM at room temperature and perform XFM.</li>
</ol>
4. Metal Imaging in the LC via XFM
<ol>
	<li>Slice example mouse brain as described above, determine LC, and measure metal levels via XFM from these brain slices containing the LC.</li>
	<li>Image elemental distributions on beamline 2-ID-E at the Advanced Photon Source (Argonne National Laboratory, Argonne IL).</li>
	<li>Record data &#8216;on the fly&#8217; as described previously31.</li>
	<li>Determine elemental concentrations in brain slices containing the LC using the program MAPS32,33.</li>
</ol>

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<li>Davies, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copper+pathology+in+vulnerable+brain+regions+in+Parkinson%27s+disease%5BTitle%5D)+AND+%22Neurobiology+of+aging%22%5BJournal%5D)">Copper pathology in vulnerable brain regions in Parkinson's disease.</a> Neurobiology of aging. 35, 858-866 (2014).</li>
<li>Davies, K. M., Mercer, J. F., Chen, N., Double, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copper+dyshomoeostasis+in+Parkinson%27s+disease%3A+implications+for+pathogenesis+and+indications+for+novel+therapeutics%5BTitle%5D)+AND+%22Clinical+science%22%5BJournal%5D)">Copper dyshomoeostasis in Parkinson's disease: implications for pathogenesis and indications for novel therapeutics.</a> Clinical science. 130, London, England. 565-574 (2016).</li>
<li>James, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+comparison+of+preparation+methodologies+for+X-ray+fluorescence+microscopy+of+brain+tissue%5BTitle%5D)+AND+%22Analytical+and+bioanalytical+chemistry%22%5BJournal%5D)">Quantitative comparison of preparation methodologies for X-ray fluorescence microscopy of brain tissue.</a> Analytical and bioanalytical chemistry. , 853-864 (2011).</li>
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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&#039;,6-diamidino-2-phenylindole)</td><td>Sigma-Aldrich</td><td>D9542-10MG</td><td></td></tr><tr><td>Dopamine &amp;beta;-hydroxylase (DBH) antibody - inhouse production (source - rabbit)</td><td>B. Eipper</td><td>-</td><td></td></tr><tr><td>Dopamine &amp;beta;-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>ASIN: B000CMFJZ2</td><td></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

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...

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 ...

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 missing the LC entirely. Thus, for one&#8217;s first time following this protocol, we recommend cutting more sections than necessary. Careful study of the brain atlas images prior to staining is very helpful. The appearance of the brainstem changes appreciably every few hundred microns and, with some experience, it is possible to know what sections are worth staining simply by the macroscopic appearance.

During the process of localizing the LC, there might be variations in the signal depending on how well the brain was oriented during sectioning. When cutting through the center of the LC, the signal is bright and covers a larger area as compared to the edge of the LC, which will show up as a signal over a much smaller area. In the case that coronal slices are slightly tilted, the LC of one side of the 4th ventricle might be apparent and the one on the other side might only be visible in an adjacent slide. Thus, one cannot always expect the appearance of both LC regions at maximum intensity within one brain slice. This artifact can be avoided by cutting the brain exactly coronal in the mouse brain slicer matrix and carefully embedding the brain into the cubical embedding mold with OCT.

Immunostaining, at least with the anti-tyrosine hydroxylase antibody, is extremely forgiving and, in our experience, works on sections up to 100 &#956;m in thickness. We have found that blocking solution is not necessary for high signal-to-noise staining of LC, reducing cost and reducing the amount of time needed to locate LC. In our experience, the staining protocol can be sped up &#8211; albeit with reduced quality and penetrance of staining &#8211; by reducing permeabilization to 2 h, primary antibody for 8 h, and secondary antibody for 2 h. Additionally, if one is simply interested in locating LC (e.g., for validating injection of a virus/tracer), sections from a fixed brain can be cut on a vibratome at 100 &#956;m thickness.

One limitation of this protocol is it, by design, requires euthanizing the animal and removing the brain. Therefore, it is not useful for in vivo localization (e.g., electrophysiologic recordings). Another limitation is that this protocol requires PFA fixation which might alter the native state of the tissue. These alterations include the elemental content such as copper, calcium, iron and zinc36. The actual alteration of metal distribution caused by PFA fixation may be tested in one sample which can be run in parallel to a non-fixed sample. A comparison of the metal distribution between these two samples will provide evidence on the effect of PFA fixation on the distribution of the metal which is of interest in a certain study. If PFA fixation must be avoided, the general principle of this protocol (locating LC by immunostaining and using adjacent sections for follow-up experiments) can be extended to frozen sections without fixation.

We note that this protocol is largely a refinement of existing methods to solve this problem. The novelty exists in tailoring previous approaches to locate a very small, easily missed nucleus. We expect that this protocol can be easily modified and extended based on need (e.g., using transgenic animals expressing fluorophores in LC to avoid immunostaining).

localization-of-the-locus-coeruleus-in-the-mouse-brain

Research

JoVE Journal

Neuroscience

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Source: Xavier, A. L. R., et al. Cannula Implantation into the Cisterna Magna of Rodents. J. Vis. Exp. (2018)This video demonstrates the procedure for implanting a cannula into the cisterna magna of an anesthetized mouse. The technique involves guiding a needle-mounted cannula through the dura into the inverted triangular cisterna magna and securing it for targeted delivery into the cerebrospinal fluid.

Cannula Implantation into the Cisterna Magna in a Mouse Model

This video demonstrates in vivo recording of local field potential (LFP) from bilateral M2 areas of the mouse brain. An anesthetized mouse is secured and prepared for the procedure. To record LFPs, insert microelectrodes into the target coordinates of the bilateral M2 areas, which are responsible for complex motor movements.

Recording of Local Field Potential from Both Hemispheres of the Mouse Brain

This protocol outlines the use of immunohistochemistry to identify locus coeruleus regions in mouse brain sections. By targeting dopamine β-hydroxylase with green fluorophore-labeled antibodies, norepinephrine-producing neurons are visualized as green fluorescence under a microscope.

Immunohistochemistry for Identifying the Locus Coeruleus Region in Mouse Brain Sections

This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Calcium Imaging in Mouse Superior Colliculus

Learn a precise protocol for localizing the locus coeruleus in mouse brain sections using immunohistochemistry and anatomical landmarks. This method enables analysis of protein expression and metal homeostasis in neurological research.

Localization of the Locus Coeruleus in the Mouse Brain

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