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. H., Torterolo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+MCH-containing+fibers+in+the+feline+brainstem:+Relevance+for+REM+sleep+regulation.">Distribution of MCH-containing fibers in the feline brainstem: Relevance for REM sleep regulation.</a> Peptides. , 50-61 (2018).</li><li>Semba, J., Toru, M., Mataga, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-four+hour+rhythms+of+norepinephrine+and+serotonin+in+nucleus+suprachiasmaticus,+raphe+nuclei,+and+locus+coeruleus+in+the+rat.">Twenty-four hour rhythms of norepinephrine and serotonin in nucleus suprachiasmaticus, raphe nuclei, and locus coeruleus in the rat.</a> Sleep. 7, 211-218 (1984).</li><li>Takeuchi, 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=Locus+coeruleus+and+dopaminergic+consolidation+of+everyday+memory.">Locus coeruleus and dopaminergic consolidation of everyday memory.</a> Nature. 537, 357-362 (2016).</li><li>Korf, J., Aghajanian, G. K., Roth, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+turnover+of+norepinephrine+in+the+rat+cerebral+cortex+during+stress:+role+of+the+locus+coeruleus.">Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of the locus coeruleus.</a> Neuropharmacology. 12, 933-938 (1973).</li><li>Sara, S. J., Segal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+sensory+responses+of+locus+coeruleus+neurons+in+the+behaving+rat:+implications+for+cognition.">Plasticity of sensory responses of locus coeruleus neurons in the behaving rat: implications for cognition.</a> Progress in brain research. 88, 571-585 (1991).</li><li>Markevich, V. A., Voronin, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+reactions+of+sensomotor+cortex+neurons+to+stimulation+of+emotionally+significant+brain+structures].">Synaptic reactions of sensomotor cortex neurons to stimulation of emotionally significant brain structures].</a> Zhurnal vysshei nervnoi deiatelnosti imeni I P Pavlova. 29, 1248-1257 (1979).</li><li>File, S. E., Deakin, J. F., Longden, A., Crow, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+the+role+of+the+locus+coeruleus+in+anxiety+and+agonistic+behaviour.">An investigation of the role of the locus coeruleus in anxiety and agonistic behaviour.</a> Brain research. 169, 411-420 (1979).</li><li>Pamphlett, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+environmental+toxicants+by+the+locus+ceruleus:+a+potential+trigger+for+neurodegenerative,+demyelinating+and+psychiatric+disorders.">Uptake of environmental toxicants by the locus ceruleus: a potential trigger for neurodegenerative, demyelinating and psychiatric disorders.</a> Medical hypotheses. 82, 97-104 (2014).</li><li>Wang, 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=Neuromelanin-sensitive+magnetic+resonance+imaging+features+of+the+substantia+nigra+and+locus+coeruleus+in+de+novo+Parkinson's+disease+and+its+phenotypes.">Neuromelanin-sensitive magnetic resonance imaging features of the substantia nigra and locus coeruleus in de novo Parkinson's disease and its phenotypes.</a> European journal of neurology. 25, 949-973 (2018).</li><li>Oliveira, L. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+dichotomy+in+spinal-+vs+prefrontal-projecting+locus+coeruleus+modules+splits+descending+noradrenergic+analgesia+from+ascending+aversion+and+anxiety+in+rats.">Functional dichotomy in spinal- vs prefrontal-projecting locus coeruleus modules splits descending noradrenergic analgesia from ascending aversion and anxiety in rats.</a> eLife. 6, (2017).</li><li>McCall, J. G., 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=CRH+Engagement+of+the+Locus+Coeruleus+Noradrenergic+System+Mediates+Stress-Induced+Anxiety.">CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety.</a> Neuron. 87, 605-620 (2015).</li><li>Borges, G. P., Mico, J. A., Neto, F. L., Berrocoso, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticotropin-Releasing+Factor+Mediates+Pain-Induced+Anxiety+through+the+ERK1/2+Signaling+Cascade+in+Locus+Coeruleus+Neurons.">Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons.</a> The international journal of neuropsychopharmacology. 18, (2015).</li><li>Simone, 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=Ethinyl+estradiol+and+levonorgestrel+alter+cognition+and+anxiety+in+rats+concurrent+with+a+decrease+in+tyrosine+hydroxylase+expression+in+the+locus+coeruleus+and+brain-derived+neurotrophic+factor+expression+in+the+hippocampus.">Ethinyl estradiol and levonorgestrel alter cognition and anxiety in rats concurrent with a decrease in tyrosine hydroxylase expression in the locus coeruleus and brain-derived neurotrophic factor expression in the hippocampus.</a> Psychoneuroendocrinology. 62, 265-278 (2015).</li><li>Carter, M. 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. G., Sinnamon, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+locus+coeruleus:+neurobiology+of+a+central+noradrenergic+nucleus.">The locus coeruleus: neurobiology of a central noradrenergic nucleus.</a> Progress in neurobiology. 9, 147-196 (1977).</li><li>Ralle, M., 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=Disease+at+a+Single+Cell+Level:+intracellular+copper+trafficking+activates+compartment-specific+responses+in+hepatocytes.">Disease at a Single Cell Level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.</a> The Journal of Biological Chemistry. 285, 30875-30883 (2010).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2013).</li><li>Bonnemaison, M. L., 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,+zinc+and+calcium:+imaging+and+quantification+in+anterior+pituitary+secretory+granules.">Copper, zinc and calcium: imaging and quantification in anterior pituitary secretory granules.</a> Metallomics : integrated biometal science. 8, 1012-1022 (2016).</li><li>Nietzold, 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=Quantifying+X-Ray+Fluorescence+Data+Using+MAPS.">Quantifying X-Ray Fluorescence Data Using MAPS.</a> Journal of visualized experiments : JoVE. , (2018).</li><li>Vogt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPS:+A+set+of+software+tools+for+analysis+and+visualization+of+3D+X-ray+fluorescence+data+sets.">MAPS: A set of software tools for analysis and visualization of 3D X-ray fluorescence data sets.</a> J. Phys. IV France. 104, 635-638 (2003).</li><li>Davies, K. M., 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+pathology+in+vulnerable+brain+regions+in+Parkinson's+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+dyshomoeostasis+in+Parkinson's+disease:+implications+for+pathogenesis+and+indications+for+novel+therapeutics.">Copper dyshomoeostasis in Parkinson's disease: implications for pathogenesis and indications for novel therapeutics.</a> Clinical science. 130, 565-574 (2016).</li><li>James, S. A., 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=Quantitative+comparison+of+preparation+methodologies+for+X-ray+fluorescence+microscopy+of+brain+tissue.">Quantitative comparison of preparation methodologies for X-ray fluorescence microscopy of brain tissue.</a> Analytical and bioanalytical chemistry. , 853-864 (2011).</li></ol>

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.

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

Research

JoVE Journal

Neuroscience

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

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

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

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.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...