Name: use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain
Uploaded: 2017-10-29T13:00:00
Description: Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

This work was supported by grants from the National Center for Research Resources (2G12RR03060-26A1); The National Institute on Minority Health and Health Disparities (8G12MD007603-27) from the National Institutes of Health; Professional Staff Congress-City University of New York (PSC-CUNY); and Faculty Research Grant (FRG II) American University of Sharjah. We thank Vidyasagar Sriramoju for introducing us to these methods.

The following protocol follows the animal care guidelines established by the Institutional Animal Care and Use Committee (IACUC) at The City College of New York, which conform to all appropriate state and federal guidelines. Anesthesia is appropriate for ferrets, and should be modified according to species studied.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56547/56547fig1.jpg" /> 
Figure 1: Flowchart outlining the major steps invol...

<ol>
	<li>Nakashima, A., Dyck, 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=Zinc+and+cortical+plasticity.">Zinc and cortical plasticity.</a> Brain Res. Rev. 59, 347-373 (2009).</li><li>Frederickson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+zinc+and+zinc-containing+neurons.">Neurobiology of zinc and zinc-containing neurons.</a> Int Rev Neurobiol. 31, 145-238 (1989).</li><li>Beaulieu, C., Dyck, R., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+glutamate+in+zinc-containing+terminals+of+the+cat+visual+cortex.">Enrichment of glutamate in zinc-containing terminals of the cat visual cortex.</a> NeuroReport. 3 (10), 861-864 (1992).</li><li>Martinez-Guijarro, F. J., Soriano, E., Del Rio, J. A., Lopez-Garcia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-positive+boutons+in+the+cerebral+cortex+of+lizards+show+glutamate+immunoreactivity.">Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity.</a> J Neurocytol. 20 (10), 834-843 (1991).</li><li>Dyck, R., Beaulieu, C., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+localization+of+synaptic+zinc+in+the+developing+cat+visual+cortex.">Histochemical localization of synaptic zinc in the developing cat visual cortex.</a> J Comp Neurol. 329 (1), 53-67 (1993).</li><li>Garrett, B., Geneser, F. A., Slomianka, L. <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+acetylcholinesterase+and+zinc+in+the+visual+cortex+of+the+mouse.">Distribution of acetylcholinesterase and zinc in the visual cortex of the mouse.</a> Anat Embryol. (Berl). 184 (5), 461-468 (1991).</li><li>Garrett, B., Osterballe, R., Slomianka, L., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoarchitecture+and+staining+for+acetylcholinesterase+and+zinc+in+the+visual+cortex+of+the+Parma+wallaby+(Macropus+parma).">Cytoarchitecture and staining for acetylcholinesterase and zinc in the visual cortex of the Parma wallaby (Macropus parma).</a> Brain Behav Evol. 43 (3), 162-172 (1994).</li><li>Dyck, R., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interdigitated+columnar+mosaic+of+cytochrome+oxidase,+zinc,+and+neurotransmitter-related+molecules+in+cat+and+monkey+visual+cortex.">An interdigitated columnar mosaic of cytochrome oxidase, zinc, and neurotransmitter-related molecules in cat and monkey visual cortex.</a> Proc. Natl. Acad. Sci. (90), 9066-9069 (1993).</li><li>Land, P. W., Akhtar, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+alteration+of+synaptic+zinc+in+rat+somatosensory+barrel+cortex.">Experience-dependent alteration of synaptic zinc in rat somatosensory barrel cortex.</a> Somatosens Mot Res. 16 (2), 139-150 (1999).</li><li>Danscher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+selenium+in+the+brain:+a+histochemical+technique+for+light+and+electron+microscopic+localization+of+catalytic+selenium+bonds.">Exogenous selenium in the brain: a histochemical technique for light and electron microscopic localization of catalytic selenium bonds.</a> Histochemistry. 76, 281-293 (1982).</li><li>Danscher, G., Howell, G., Perez-Clausell, J., Hertel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dithizone,+Timm's+sulphide+silver+and+the+selenium+methods+demonstrate+a+chelatable+pool+of+zinc+in+CNS:+a+proton+activation+(PIXE)+analysis+of+carbon+tetrachloride+extracts+from+rat+brains+and+spinal+cords+intravitall+treated+with+dithizone.">The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS: a proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitall treated with dithizone.</a> Histochemistry. 83, 419-422 (1985).</li><li>Gallyas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+myelin+by+means+of+physical+development.">Silver staining of myelin by means of physical development.</a> Neurol Res. 1 (2), 203-209 (1979).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Res. 171 (1), 11-28 (1979).</li><li>Mir&#243;-Berni&#233;, N., Ichinohe, N., Perez-Clausell, J., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-rich+transient+vertical+modules+in+the+rat+retrosplenial+cortex+during+postnatal+development.">Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development.</a> J Neurosci. 138 (2), 523-535 (2006).</li><li>Ichinohe, N., Rockland, K. S. <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+synaptic+zinc+in+the+macaque+monkey+amygdala.">Distribution of synaptic zinc in the macaque monkey amygdala.</a> J Comp Neurol. 489 (2), 135-147 (2005).</li><li>Innocenti, G. M., Manger, P. R., Masiello, I., Colin, I., Tettoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+callosal+connections+of+visual+areas+17,+18,+19+and+21+in+the+ferret+(Mustela+putorius).">Architecture and callosal connections of visual areas 17, 18, 19 and 21 in the ferret (Mustela putorius).</a> Cereb Cortex. 12 (4), 411-422 (2002).</li><li>Khalil, R., Levitt, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+histochemistry+reveals+circuit+refinement+and+distinguishes+visual+areas+in+the+developing+ferret+cerebral+cortex.">Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex.</a> Brain Struct Funct. 218, 1293-1306 (2013).</li><li>Manger, P. R., Masiello, I., Innocenti, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Areal+organization+of+the+posterior+parietal+cortex+of+the+ferret+(Mustela+putorius).">Areal organization of the posterior parietal cortex of the ferret (Mustela putorius).</a> Cereb Cortex. 12, 1280-1297 (2002).</li><li>Wong, P., Kaas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectonic+subdivisions+of+neocortex+in+the+gray+squirrel+(Sciurus+carolinensis.).">Architectonic subdivisions of neocortex in the gray squirrel (Sciurus carolinensis.).</a> The anatomical record. 291, 1301-1333 (2008).</li><li>Land, P. W., Shamalla-Hannah, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+zinc-containing+cortical+circuits+during+a+critical+period+of+postnatal+development.">Experience-dependent plasticity of zinc-containing cortical circuits during a critical period of postnatal development.</a> J Comp Neurol. 447 (1), 43-56 (2002).</li><li>Czupryn, A., Skangiel-Kramska, J. <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+synaptic+zinc+in+the+developing+mouse+somatosensory+barrel+cortex.">Distribution of synaptic zinc in the developing mouse somatosensory barrel cortex.</a> J Comp Neurol. 386, 652-660 (1997).</li><li>Timm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Histochemie+der+Schwermetalle.+Das+Sulfid-Silber-Verfahren.">Zur Histochemie der Schwermetalle. Das Sulfid-Silber-Verfahren.</a> Dtsch Z ges gerichtl Med. 46, 706-711 (1958).</li></ol>

The current study employs a histochemical technique based on a modified version of the Danscher (1982) method10, whereby synaptic zinc localization may be detected and visualized in the brain. This method essentially works by injecting the animal with the zinc chelator sodium selenite (Na2SeO3) (15 mg/kg). Following injection, the selenite travels to the brain and binds to free zinc that is localized to presynaptic vesicles of zinc containing neurons. Zinc ions bound to molec...

Histological stains have traditionally been used to aid in the identification of cortical areas in various species by revealing differences in architectonic features. The combined use of histochemical techniques such as for Nissl substance, cytochrome oxidase (CO) reactivity, or myelin can prove fruitful as they reveal similar areal boundaries in the adult brain. However, these histochemical stains do not always adequately reveal clear boundaries between cortical areas and layers in the immature brain.
In the central nervous system, zinc has several critical functions that include stabilizing DNA structure, acting as an enzyme cofactor, participating in numerous regulatory functions, and acting as a neuromodulator through its presence in synaptic vesicles1. Synaptic zinc is unique as it can be visualized using histological methods, whereas protein-bound zinc cannot be visualized2. This feature has been exploited to reveal the synaptic zinc pattern in different cortical regions, and synaptic zinc histochemistry has been used in a number of studies. A subset of glutamatergic neurons in the cerebral cortex contain zinc in the presynaptic vesicles within their axon terminals3,4. Histochemical studies have revealed a heterogeneous distribution of synaptic zinc in the cerebral cortex5,6,7. There appears to be a different areal and laminar distribution of histochemically reactive zinc in different cortical regions (e.g., visual versus somatosensory cortex), or layers (e.g., zinc levels in the supragranular and infragranular layers of primary visual cortex are substantially higher than in thalamocortical input layer IV with relatively low synaptic zinc levels)5,8,9. The heterogeneity in synaptic zinc staining observed in the cortex is especially advantageous as it facilitates areal and laminar identification.
Here we present a detailed description of a synaptic zinc histochemical procedure, which is a modified version of Danscher&#39;s 1982 method10. This method utilizes selenite injected intraperitoneally (IP) into animals as a chelating agent. The selenite travels to the brain to react with pools of free zinc found in vesicles of a subset of glutamatergic synapses in the brain. This reaction yields a precipitate that can be enhanced subsequently by silver development2,10,11.
This procedure reveals laminar and areal patterns of synaptic zinc staining; densitometric analysis may be used to assess these patterns both qualitatively and quantitatively in the adult and immature brain to study effects of other interventions, such as sensory, environmental, pharmacological, or genetic manipulations. Moreover, one may also want to assess potential developmental changes in the distribution of synaptic zinc in other cortical or subcortical structures in other model systems. The quantitative information that densitometric analysis provides in this method can be advantageous for following brain development over time. This protocol provides a companion to other immuno- and histochemical markers to reveal laminar and areal boundaries.

<table>
	<tbody>
		<tr>
			<td>Euthasol (Euthanasia solution)</td>
			<td>Henry Schein</td>
			<td>710101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma-Aldrich</td>
			<td>214485</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Ketaved)</td>
			<td>Henry Schein</td>
			<td>48858</td>
			<td>100 mg/ml injectables</td>
		</tr>
		<tr>
			<td>Xylazine (Anased)</td>
			<td>Henry Schein</td>
			<td>33198</td>
			<td>100 mg/ml injectables</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma-Aldrich</td>
			<td>G9752-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>W302600</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma-Aldrich</td>
			<td>H9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver lactate</td>
			<td>Sigma-Aldrich</td>
			<td>85210</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish gelatine</td>
			<td>Sigma-Aldrich</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td>(Type III, from equine heart)</td>
		</tr>
		<tr>
			<td>Catalse</td>
			<td>Sigma-Aldrich</td>
			<td>C10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Domino</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used for dehydration prior to slide mounting</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Brain Research Laboratories</td>
			<td>#3660-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted unsubbed slides</td>
			<td>Brain Research Laboratories</td>
			<td>#3875-FR</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>American Optical Company</td>
			<td>860</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Adope Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
			<td></td>
			<td>For densometric measurements</td>
		</tr>
		<tr>
			<td>Plastic tray</td>
			<td>Any standard plastic tray may be used</td>
			<td></td>
			<td>to immerse slides in developer solution</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Any standard hotplate may be used</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

The major steps involved in this protocol to stain brain sections for synaptic zinc are presented in a flowchart in Figure 1. The protocol can be divided into three phases: 1) Perfusion and tissue collection, 2) Tissue preparation and staining, and 3) Zinc histochemistry. Briefly, during the first phase of the protocol, the animal is anesthetized and injected IP with the appropriate dose of sodium selenite. After a sufficient time period (ideally 60 - 90 min)...

Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

The overall goal of this method is perform histochemical staining on ferret frozen brain sections to illustrate utilization of synaptic Zinc staining patterns in distinguishing different regions and layers in the developing and adult brain. This method can help answer key questions such as the qualitative and quantitative differences in synaptic Zinc staining pattern among different brain regions and layers. The main advantage is that this technique is simple and reliable allowing one to identify different regions and layers in the developing and adult brain based on unique staining patterns.Although this method has been utilized to provide insight in demarcating areas and layers in the feral visual cortex it can also be used to differentiate layers in different brain regions of rodents, cats, or primates. To prepare 200 milliliters of the developer's solution, add 40 grams of gum arabic slowly to 120 milliliters of hot water and keep stirring it with a glass rod to dissolve completely. Then it let it cool for few minutes, then filter it through six to eight layers of gauze cloth in a funnel.Next add 5.04 grams citric acid and 4.7 grams sodium citrate in 20 milliliters of distilled water and dissolve to make the citrate buffer. Then add 1.7 grams hydroquinone to 30 milliliters of heated distilled water. Add 0.22 grams silver lactate to 30 milliliters of water to make silver lactate solution.In the following order, mix the gum arabic, citrate buffer, hydroquinone, and finally, silver lactate solutions to prepare the developer's solution and keep it in the dark. To start this procedure, inject a dose of 15 milligrams per kilogram body weight of sodium selenite solution, a Zinc chelator, intraperitoneally into the anesthetized ferret. Then wait for 60 to 90 minutes for the sodium selenite solution to reach the brain.Then inject an overdose of 100 milligrams per kilogram phenobarbital intraperitoneally to euthanize the animal and wait for several minutes for euthasol to work. Perform transcardial perfusion on the euthanized ferret first with normal saline solution for one minute, and then with 4%paraformaldehyde for 20 minutes. Then use a fixative with sucrose to complete the total fixation procedure in one hour.Use a scalpel to make a mid-line incision starting from the nose to neck to expose the skull. Delicately remove the brain and then use a scalpel blade to separate the hemispheres. Then cut and separate the posterior part from the rest of the brain and post-fix for two to four hours.Let the brain sink by keeping the tissue in 30%sucrose solution in 0.1 molar phosphate buffer. After the brain sinks, use a freezing sliding microtome to make semi-tangential 40 micrometer thick sections through the visual cortex. Use a paintbrush to collect all the brain sections and place them in a tackle box filled with phosphate buffered saline.Then, quickly mount one or two series of brain sections on egg-white coated slides. Keep the slides at room temperature overnight to dry. After the slides have dried, immerse them in absolute alcohol for 15 minutes.Then wait an hour to let the slides dry completely at room temperature. Quickly submerge the sections in 1%gelatin solution for 10 seconds then drain carefully. Keep the sections at room temperature to dry overnight.Use a plastic tray to arrange the slides side by side. Then pour over the developing solution and make sure that the slides are completely submerged in it. Remember to check the sections every 30 minutes for the development of the reaction.If the slides have been stained appropriately, remove the slide-mounted sections from the tray and keep the slides on a slide rack. Use a large glass-staining dish to wash the slides inside the slide rack under warm running water for 10 minutes to wash the gelatin coat and the outer silver deposit. After drying the slides at room temperature, dehydrate them for five minutes in 100%ethanol and clear for five minutes in xylene.Finally, place a cover slip with mounting media on the slide. Use a bright-field microscope to examine the sections and take pictures of the areas of interest. Select Zinc stained sections from photomicrographs of regions of interest to randomly choose cortical columns of approximately 450 micrometer width.In each region of interest, choose an appropriate number of sample columns from several different brain sections. Then transfer the column images to an image processing software. In the software, use the rectangular section tool to encompass the entire cortical column.Next utilize the invert tool to contrast reverse the image. Then use the plot profile tool to make a two-dimensional graph of the pixel intensities along the line to produce optical density profiles from the images. Finally, use the plot profile options to convert the plot profile graph to a vertical profile.Then click on plot profile once more. Photomicrographs of semi-tangential Zinc stained sections of juvenile ferret brain show the characterization of over-stained and under-stained tissues. Over-stained tissue develops very dark brown color with inadequate laminar variation and densely stained white matter.In contrast, under-stained tissues develop light-brown color with minimally stained white matter. Photomicrographs of semi-tangential sections show synaptic Zinc in cytochrome oxidase staining in visual cortical areas of an adult ferret. Synaptic Zinc staining intensity is high in the supragranular and infragranular layers of areas 17 and 18, while layer four stains lightly.Conversely, layer four, of area of 17 and 18, is heavily stained when stained for cytochrome oxidase reactivity. Columnar photomicrographs show laminar distribution of synaptic Zinc in all the cortical layers with their complementary normalized optical density values. To reflect changes in synaptic Zinc staining according to cortical depth, normalized plots of optical density values were generated at each visual cortical area.The trough present in plot profiles shows low synaptic Zinc staining in layer four of areas 17 and 18. This procedure can help explain if there are any developmental changes and staining intensity in particular brain regions over time. This technique proved useful for neuroanatomy researchers to explore how sensory, environmental, pharmacological, or genetic manipulations may affect the distribution of synaptic Zinc staining in the adult and the immature brain in a range of species.Thanks for watching. Good luck with your experiments.

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Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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 ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...