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

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.

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