This work was supported by Humboldt Universit&#228;t zu Berlin, the Bernstein Center for Computational Neuroscience Berlin, the German Center for Neurodegenerative Diseases (DZNE), the German Federal Ministry of Education and Research (BMBF, F&#246;rderkennzeichen 01GQ1001A), NeuroCure, and the Gottfried Wilhelm Leibniz prize of the DFG. We thank Shimpei Ishiyama for excellent graphic design and Juliane Diederichs for excellent technical assistance.

All experimental procedures were performed according to the German guidelines on animal welfare under the supervision of local ethics committees (LaGeSo). Human and bat brain data were derived from Naumann et al.5 The following procedure is performed on a male adult Wistar rat (strain: RJHan:WI).
1. Perfusion and Brain Extraction
NOTE: In order to obtain a homogenously fixed and blood-free brain, transcardial perfusion of the animal is...

<ol>
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Modularity in the cerebral cortex has been identified using a variety of techniques. The earliest studies typically identified cortical modules by either visualizing cell dense regions, or an absence of fibers1. Subsequent methods have utilized the presence of dendritic bundles24, afferents from a particular region25, or enrichment of neurotransmitters26. Here we demonstrate two techniques, (i) cytochrome oxidase histochemistr...

Some of the most significant changes in the brain structure across phylogeny can be observed in the cerebral cortex. Despite significant differences, the cortex of animals follows a common pattern and can be broadly divided in two distinct ways, by layers and areas1. Cortical layers lie parallel to the surface of the brain and vary in number from 3 layers in reptilian cortices2 to 6 layers in mammalian cortices1. Cortical areas on the other hand are distinct regions of the cortex which largely correspond to distinct functionalities, e.g., the somatosensory cortex is involved in the sensation of touch or the visual cortex in processing visual inputs. These cortical areas can often be subdivided into patches or modules3, which are regularly repeating anatomical structures, essentially found parallel to the pial surface of the brain. Cortical modules may be confined to a particular layer4, or extend across several layers5.
Standard sectioning methods of the brain involve sections normal to the surface of the brain, like coronal or sagittal. While these methods can be used to visualize cortical modules, a multitude of interesting features can be revealed when the cortical modules are visualized tangentially, in a plane parallel to the surface of the brain. For instance, somatosensory modules in the rodent brain representing whiskers, appear as barrels when visualized normal to the brain surface, and thus the regions derive the name barrel cortex. However, on visualizing the barrels in a tangential orientation, they reveal a whisker-map, with the barrels being laid out in a topographic orientation mirroring the exact layout of the whiskers on the external body surface. In certain cases, modular arrangement has even escaped detection for considerable periods, when visualized in a non-tangential manner. The medial entorhinal cortex, is known for the presence of grid cells, neurons which fire in a regular hexagonal pattern when an animal is traversing an environment. Even though it is a heavily investigated area, until recently, the presence of patches or modules of cells in the medial entorhinal cortex, which are physically laid out in a hexagonal pattern6, had escaped detection. The presence and arrangement of these modules, in the rat brain, was facilitated by making tangential sections of the medial entorhinal cortex and investigating the cytoarchitecture in a layer-wise manner.
Subsequent to sectioning, the particular aspect of visualization of cortical modules can also be realized in multiple ways. Classically, studies have delineated modules based on cell density or fiber layout1. Another popular approach is the use of cytochrome oxidase histochemistry, which reveals areas of higher activity8. Newer approaches include looking at genetically determined cell types, distinguished on the basis of their protein expression profiles6,8.
In this study, we highlight methods to isolate the cortex from mammalian brains, obtain flattened tangential sections, and visualize cortical modules based on cytochrome oxidase histochemistry and immunohistochemistry of cell-type specific proteins.

<table>
	<tbody>
		<tr>
			<td>Cytochrome oxidase staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;Diaminobenzidine tetrahydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5637</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Saccharose</td>
			<td>Carl Roth&#160;</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium nickel(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A1827</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antigen retrieval</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phosphate buffer/phosphate-buffered saline/prefix/PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-Sodium hydrogen phosphate dihydrate</td>
			<td>Carl Roth</td>
			<td>4984.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON-X 100</td>
			<td>Carl Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k puriefied from chicken gut, Mouse monoclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000347</td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k from recombinant rat calbindin D-28k, Rabbit polyclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000340</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V, biotin free</td>
			<td>Carl Roth</td>
			<td>0163.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mounting or freezing media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount (immunofluorescence)</td>
			<td>Sigma-Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt (histochemistry)</td>
			<td>Sigma-Aldrich</td>
			<td>03989</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium</td>
			<td>Leica Biosystems</td>
			<td>NC0696746</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Alcohol dehydration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Carl Roth</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth</td>
			<td>P075.3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth</td>
			<td>6752.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>Fluka</td>
			<td>78475</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Devices/tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microm HM 650V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jung RM2035</td>
			<td>Leica Biosystems</td>
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visualization of cortical modules in flattened mammalian cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

We obtained flattened cortical sections of the somatosensory cortex in a variety of brains, and processed them for cytochrome oxidase histochemistry to visualize the somatotopic modules representing different body parts. This comparative approach allows studying the evolutionary forces that shape cortex, e.g., showing highly conserved representation of mystacial vibrissae in rodents and lagomorpha as barrels21 (Figure 2). In c...

Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

Visualization of Cortical Modules in Flattened Mammalian Cortices

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

The overall goal for this procedure for cortex flattening and staining is to visualize cellular modules in the cortical regions in a layer-wise manner. This method can answer key questions in neuroscience, like how cortical structure spreads into the brain, and how it might relate to function. The main advantage of this technicque is you can compare cortical architecture in a laminal fashion, and compare various aspects of it, like how it's conserved the class evolution, or how it varies in function.Demonstrating the technique will be Simon Lauer, and Undine Schneeweib, a student and a technician from the lab of Professor Michelle Black. To begin, transcardially perfuse the animal to obtain a homogeneously fixed and blood-free brain. After removing the brain, submerge it in 0.1 molar phosphate buffer.If sections with large cortical areas are desired, flatten the brain prior to further postfixation. However, to obtain sections of harder-to-reach areas, like the medial entorhinal cortex, postfix the brain in 4%PFA for 24 hours before flattening. Prepare to keep the brain moist with buffer during the whole dissection.To begin, separate the hemispheres using a scalpel. If the brain has not been postfixed, be careful when handling it. Next, while securing the brain by the cerebellum, gently insert the spatula to open a dissection plane in the corpus callosum.The round tip of spatula should point away from the cortex. Then, manipulate the spatula to divide the thalamus and cortex. Next, separate the subcortical structures using a razor blade.Next, separate the subcortical structures using a scalpel or the edge of the spatula. To improve the flattening, make a clean cut through the striatum, nucleus accumbens, and orbital frontal cortex, since they increase the thickness of the cortex in the lateral region. Then add a relieving cut at the base of the inner region of prefrontal cortex.This will allow for unfolding of medial portions of the cortex. Now, place a hemisphere cortex down on a glass slide. Then, place two clay cylinders on either side of the tissue.The clay must a 10 to 20%thinner than the brain tissue, as the clay defines the extent of flattening. Next, place a glass side tangentially onto the cortex, contacting the lateral portion first. Then, set a weight on the glass, with the mass centered over the lateral portion.Now, let the assembly sit for three to five hours in phosphate buffer at four degrees Celsius. The real key to this preparation is to get the brain as flat as possible, as this has the greatest impact on viewing the cortical map. Lastly, if needed, postfix the brain.Transfer the flattened hemisphere to 1%PFA, or 2%PFA if it was not taken from a perfused animal. Then, let it fix for 24 hour at 4 degrees Celsius, with gentle agitation. First, wash the flattened hemisphere in phosphate buffer for 15 minutes.Then, slice the hemisphere tangentially on a Vibratome. Fix the tissue in position using a small volume of cyanoacrylate and gentle pressing. Be careful, as too much glue can lead to cutting artifacts.Now, cut the hemisphere from the thicker and more stable end towards the thinner end at the required thickness. If low fixative concentrations were used, cut the tissue slowly with a high amplitude. Next, wash the sectioned tissue in hepes buffer for 15 minutes with gentle agitation.During the wash, prepare fresh cytochrome oxidase staining solution. And add the DAB component just before using it. Now, incubate the sections in the freshly made staining solution at room temperature with shaker and keep watch.Depending on the amount of fixation, staining could be observable in 10 minutes or it make take several hours. If the reaction is going too slow, increase the temperature to 37 degrees Celsius. To stop the reaction, transfer the sections to a bath of 4%PFA and let them incubate for 15 minutes with gentle agitation.Then, wash the sections three times, with hepes buffer for 10 minutes per wash. Now, mount and dry the sections on glass slides. And dehydrate them using progressively stronger ethanol baths.Then, wash the slides in Isopropanol for five minutes, followed by Xylene for five minutes. After the Xylene bath, immediately add a quick-hardening, non water-based mounting medium. Water-based mediums will degrade the stain.After adding a cover slip, store the slides at four degrees Celsius until imaged. Cortical sections of the somatosensory cortex in a variety of brain were processed, using the described procedures. This comparative approach allows for the study of the evolutionary forces that shape cortex, such as the highly conserved representation of mistacial vibrissaer in rodents and lagomorpha as barrels.The architecture of the medial entorhinal cortex was observed sections parallel to the peel surface, using tangential sectioning of the medical entorhinal cortex in mice, rats, and Egyptian fruit bats. In addition, human brains were processed using gentle flattening of the cortex and tangential slices. All of the brains were cryopreserved, and sectioned on a cryostat at 60 microns.Immunostaining was used to label calbindin-positive parameatal cell modules. In the entorhinal cortex the calbindin modules show a remarkable periodicity in each species, and vary in size by only a factor of 10, across a 20, 000-fold variation in brain size. After watching this video you should have a good understanding of how to obtain tangential sections for analysis.Once mastered, this technique can be completed in a couple of hours. While attempting this procedure, it's important to remember to flatten the brain properly, instead the size, the quality, or the final results. Don't forget that working with the agents like PFA and DAB can be extremely hazardous, and you should always wear protective clothing and work under the field light.

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12.6K 조회수.  Humboldt University of Berlin. The overall goal for this procedure for cortex flattening and staining is to visualize cellular modules in the cortical regions in a layer-wise manner. This method can answer key questions in neuroscience, like how cortical structure spreads into the brain, and how it might relate to function. The main advantage of this technicque is you can compare cortical architecture in a laminal fashion, and compare various aspects of it, like how it's conserved the class evolution, or how it varies in f...