Name: visualization of cortical modules in flattened mammalian cortices
Uploaded: 2018-01-22T15:00:00.000+00:00
Duration: 8 min 49 s
Description: Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

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 highly encouraged, as residual blood increases unspecific background signal during staining. Nevertheless, it is also possible to obtain flattened sections from un-perfused specimen and stain them. The ease of handling the specimen varies with the concentration of fixative used. Too little fixation increases the risk of handling damage to the brain during flattening and cutting, while too high concentrations lower the flexibility for flattening and the quality of the staining signal.
<ol>
	<li>Transcardially perfuse the animal, using a 23G needle. For a more detailed guide, see Gage et al.9
	<ol>
		<li>Use phosphate buffer saline10 (PBS; 0.02 M; pH 7.4) to flush out blood from the brain and body of the animal. Continue until infusion liquid appears clear, with at least 150 mL PBS being infused through the vascular system. 
		NOTE: Best results are achieved at a pressure similar to the range of the physiological blood pressure of the animal being perfused (e.g., rat: 120-130 mmHg). Optionally, add heparin (10 U/mL) to the solution to avoid blood coagulation11.</li>
		<li>To fix the brain, transcardially infuse a minimum of 100 mL paraformaldehyde12 (PFA, 4%; in 0.1 M phosphate buffer (PB)13) until the neck of the animal is stiff. 
		CAUTION: PFA is a potential carcinogen. 
		NOTE: Best results for histochemical activity staining, such as for cytochrome oxidase, are achieved, when using 2% PFA. For other staining, such as immunohistochemistry, 4% PFA is preferred.</li>
	</ol>
	</li>
	<li>Extract the brain from the skull. 
	NOTE: See Gage et al.9 for details.
	<ol>
		<li>Isolate the head using large scissors or bone shears.</li>
		<li>Using fine scissors, cut the skin along the midline from neck to nose. Pull apart the skin to expose the skull. Remove the neck and temporal muscles using fine scissors.</li>
		<li>Make a midline cut through the interparietal bone starting from the foramen magnum up to the parietal bone.</li>
		<li>Use Rongeurs to peel off the interparietal bone. Slide fine scissors along the sagittal suture and cut from the posterior end of the parietal to the anterior end of the frontal bones.</li>
		<li>Use Rongeurs to peel off the parietal and frontal bones. Alternatively, lift the bones away using forceps. Use a spoon to cut ventral nerve cords and remove the brain. Transfer the brain to PB (0.1 M; pH 7.4).</li>
	</ol>
	</li>
	<li>Fix un-perfused brains by immersion in 4% PFA for 1-3 h at 4 &#176;C (depending on the brain size and probe: Shrew: ~ 1 h, Human: ~ 3 h). 
	NOTE: For large and gyrencephalic brains like humans, isolate and cut out the area of interest to reduce fixation time.</li>
	<li>To obtain larger cortical areas in a section, flatten the brain prior to further post-fixation; proceed to step 2. However, to obtain sections of harder to reach areas like the medial entorhinal cortex, post-fix the brain in 4% PFA for 24 h before proceeding to step 2.</li>
</ol>
2. Brain Dissection and Flattening
<ol>
	<li>Separate the brain hemispheres by using a razor blade (lissencephalic brains) or scalpel (gyrencephalic brains). 
	NOTE: If the brain has not been post-fixed, it is susceptible to damage by handling.</li>
	<li>Optional step for aiding subsequent section registration and shrinkage estimation:
	<ol>
		<li>Puncture the cortex in an area of non-interest, with a 35G needle normal to the cortical surface. Repeat the step two times at defined distances along the cortical sheet. To determine the distance along the cortical sheet, use a pre-cut thread at a fixed length (e.g., 5 mm) and lay it along the cortical surface to determine points of puncture.</li>
	</ol>
	</li>
	<li>Lissencephalic brains: Remove subcortical structures by using a dissecting spatula. Critical: Keep the brain moist with PB (0.1 M; pH 7.4) throughout the whole procedure.
	<ol>
		<li>Hold the brain by the cerebellum and gently insert the spatula to open a dissection plane in the corpus callosum. The round tip of spatula should point away from the cortex.</li>
		<li>Slide the spatula further and pull gently until the spatula is between the thalamus and cortex. Separate subcortical structures with scratching motions.</li>
		<li>Inspect the hemisphere for even thickness. 
		NOTE: Any uneven regions might result in a trans-layer gradient during sectioning. For tangential sectioning, use a scalpel to make a clean cut through the brain, parallel to the pial surface of the region from which tangential sections are desired.</li>
		<li>Optional steps to improve flattening quality:
		<ol>
			<li>Make a clean cut through the striatum, nucleus accumbens, and orbitofrontal cortex, since they increase the thickness of the cortex in the lateral region. Also, add a relieving cut at the base of the inner region of prefrontal cortex, which allows unfolding of medial portions of the cortex.</li>
		</ol>
		</li>
		<li>Place the hemisphere (cortex facing down) on a glass slide. Proceed to step 2.6.</li>
	</ol>
	</li>
	<li>Gyrencephalic brains: Remove white matter to unfold gyri (for detailed protocols, see: Sincich et al.14; Tootell and Silverman15). Critical: Use PB (0.1 M; pH 7.4) to keep the brain moist at all time.
	<ol>
		<li>Put the area of interest on a damp filter paper in a large Petri dish, with the cortex facing up.</li>
		<li>Remove the arachnoid membrane and pia using two forceps.</li>
		<li>Use a damp cotton swab and insert gently in each sulcus to detach adhesions between gyri.</li>
		<li>Turn the brain around by using another damp filter paper.</li>
		<li>Use two curved micro spatulas to unfold single gyri. Use the convex side of the spatulas to tease apart the white matter until reaching close to the concave end of the gyrus. Use a damp cotton swab and proceed with swirling motions to reach the concave end of the gyrus.</li>
		<li>Tease apart single gyri. If necessary, add small relieving cuts if the tension is too high.</li>
		<li>Flatten the unfolded brain in the Petri dish or a similar container.</li>
	</ol>
	</li>
	<li>Optionally, place larger brains on a filter paper covered sponge, to ensure that regions do not dry out.</li>
	<li>Place two rolled pieces of clay on both sides. Critical: As the thickness of the clay defines how much the hemisphere can be flattened, ensure that the clay is 10-20% thinner than the un-flattened cortex.</li>
	<li>Gently press a second glass slide/small Petri dish on the cortex until the hemisphere is fully flat. To obtain the best results for a desired region, place the glass slide first on the respective area. To obtain the best results for lissencephalic brains, place the glass slide tangentially at the lateral portion first and apply concentrated pressure on this region.</li>
	<li>Put a weight (e.g., a ceramic watch glass) on the glass slides/Petri dish and flatten the hemisphere for 3-5 h at 4 &#176;C in PB.</li>
	<li>For post-fixation, release the pressure and remove the glass slides. Put the flattened hemisphere in PFA (free floating) for 24 h on a shaker at 4 &#176;C. 
	NOTE: Best results for histochemical activity stainings are achieved when using 1% PFA. For other stainings, such as immunohistochemistry, for un-perfused brains, 2% PFA can be used.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56992/56992fig1.jpg" /> 
Figure 1: Schematic representation of workflow for flattening of a rat cortical hemisphere and visualization of modules in somatosensory cortex. Subsequent to the transcardial perfusion, the brain of a mouse was dissected (A). Subcortical structures were removed and cortex was flattened between two glass slides in phosphate buffer (B). Flattened hemisphere (C) was post-fixed, tangentially sectioned, and stained for cytochrome oxidase activity (D). Scale bars = 1 cm. R: Rostral, C: Caudal, L: Lateral, M: Medial. Figure adapted from Lauer et al.23 <a href="//cloudfront-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56992/56992fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Cutting Tangential Sections
NOTE: Depending on the requirements of the staining protocols, the cutting procedure and thickness can be adapted. A vibratome was used to cut the hemispheres for further histochemical processing (step 3.2) at 80-150 &#181;m. However, for immunohistochemical processing, thinner sections are desired and a freezing microtome was used for sectioning (step 3.3) at 10-60 &#181;m. See Video 1.
<ol>
	<li>Wash the flattened hemisphere in PB (0.1M; pH 7.4) for 15 min.</li>
	<li>Cut the hemisphere on a vibratome.
	<ol>
		<li>Place the hemisphere tangentially on the slicing holder. Optionally, press again gently with a glass slide before fixing it in position (e.g., with superglue). 
		NOTE: Minimize the amount of glue used, because cutting artifacts could result due to the small thickness of flattened hemispheres.</li>
		<li>Cut the hemisphere from the thicker and more stable end towards the thinner end (posterior to anterior, here) at the required thickness. Proceed to step 3.4. 
		NOTE: If low fixation concentrations were used, cut it at a slow speed and a high amplitude.</li>
	</ol>
	</li>
	<li>Cut the hemisphere on a freezing microtome.
	<ol>
		<li>Cryoprotect the brain by immersing it in a 30% sucrose solution (in PB) until it sinks. 
		NOTE: Depending on the size of the tissue being cryoprotected, sinking in the solution can range from a few hours to a few days. For larger brains alternative cryoprotection methods might be considered (see Rosene et al.16).</li>
		<li>Form an ice base on the freezing microtome to mount the brain. Construct the ice base by freezing PB on the block face of the freezing microtome. Critical: Ensure that the surface of the ice base is parallel to the microtome blade. To do this, cut the empty ice base using the microtome blade to exactly align the ice base parallel to the cutting surface.</li>
		<li>Embed the brain in freezing medium and mount it to the block face of the microtome with the area of interest parallel to the block face. Face the pial surface of the brain towards the block face of the microtome. Critical: Adjust the freezing temperature depending on sample size; higher temperatures ensure better section integrity while sectioning, but larger sections require lower temperatures to freeze sample uniformly.</li>
		<li>Cut the brain tangentially at the required thickness (slower and uniform cutting speeds result in best section quality).</li>
	</ol>
	</li>
	<li>Wash the sections in PB for 15 min on a shaker.</li>
</ol>
<img alt="Video Snapshot" src="/files/ftp_upload/56992/56992video1.jpg" /> 
Video 1: Schematic video of tangential sectioning from a rat medial entorhinal cortex and layout of parasubicular and entorhinal modules. The medial entorhinal cortex of a rodent brain is situated at the posterior end of the cortex and is tilted towards the medial and ventral side. Tangential sections are obtained by orienting a knife along this angle. Consequently, appropriate cell-type specific staining reveals modular structures in the medial entorhinal cortex and adjoining parasubiculum. Video adapted from Ray and Brecht8. <a href="https://cloudfront-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56992/Video_1_Tangential_Procedure_MEC.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
4. Visualization of Cortical Modules Using Cytochrome Oxidase Staining
NOTE: Different staining protocols have been developed for histochemical detection of cytochrome oxidase activity, e.g., first by Wong-Riley17 and later modified by Divac et al.18 This protocol is based on the one by Divac et al.18, since the use of nickel-ammonium sulfate (NiAS) results in a higher contrast and better defined modules in stained cortical areas.
<ol>
	<li>Prepare the cytochrome oxidase staining solution (see Divac et al.18). For 10 mL of solution, add: 10 mL HEPES buffer (0.1 M, pH 7.4), 400 mg sucrose, 12.5 mg NiAS, 2 mg cytochrome C, 6 mg diaminobenzidine (DAB). 
	CAUTION: DAB and NiAS are carcinogenic. 
	NOTE: Add DAB just prior to incubation of sections.</li>
	<li>Wash the sections in HEPES buffer (0.1 M, pH 7.4) on a shaker for 15 min.</li>
	<li>Incubate the sections in the staining solution at room temperature on a shaker. 
	NOTE: Observe the speed of the staining. If there is no visible reaction, change to incubation at 37 &#176;C. Depending on the amount of fixation, staining can be observed after 10 min or in several hours.</li>
	<li>Stop the reaction by adding 4% PFA; this prevents unwanted re-dying and increase of background signal.</li>
	<li>Wash the sections three times using HEPES buffer for 10 min.</li>
	<li>Mount and dry the sections on glass slides.</li>
	<li>Dehydrate the sections with an increasing alcohol row:
	<ol>
		<li>Wash the slides in 60% ethanol for 1 min. Wash the slides in 80% ethanol for 1 min. Wash the slides in 96% ethanol for 2 min. Wash the slides in 100% ethanol for 3 min. Wash the slides in isopropanol for 5 min. Wash the slides in xylene for 5 min.</li>
	</ol>
	</li>
	<li>Immediately add a quick hardening-mounting medium, and add a coverslip. Critical: Do not use water-based mounting mediums, as NiAS will be washed out and degrade the staining.</li>
	<li>Keep the sections at 4 &#176;C for long term storage.</li>
</ol>
5. Visualization of Cortical Modules Using Immunohistochemical Staining
NOTE: Multiple protocols are available for immunohistochemistry, optimized for the specimen and the type of probe. Adaptations can be made as required, by varying concentrations of antibodies, permeabilizing agents, and incubation times. The following protocol leads to good results for detecting a large range of antibodies and visualization by fluorescent probes.
<ol>
	<li>Wash the sections in PBS (0.1 M; pH 7.4) for 15 min.</li>
	<li>Optional: Perform antigen retrieval to unmask an epitope using a water bath (based on Jiao et al.19; for alternative methods, see Pileri et al.20).
	<ol>
		<li>Preheat a water bath to 80 &#176;C. Prepare tri-sodium citrate buffer19 (pH 8.0) and preheat it in the water bath to 80 &#176;C.</li>
		<li>Transfer the sections to tri-sodium citrate buffer. Incubate the sections for 30 min at 80 &#176;C</li>
		<li>Cool the sections down to room temperature</li>
		<li>Wash the sections in PBS for 15 min.</li>
	</ol>
	</li>
	<li>Wash the sections two times in PBS-X (0.5% Triton-X, in 0.1 M PBS) for 15 min.</li>
	<li>Block unspecific epitopes by incubating the sections in a solution of Bovine Serum Albumin (BSA; 2.5%) and Triton-X (0.75%) in PBS for 2 h.</li>
	<li>Incubate the sections in primary antibody, e.g., Calbindin- D28k (1:5,000, 1% BSA in PBS-X), for 2-3 days on a shaker at 4 &#176;C. 
	NOTE: Optimal dilution for the primary antibody depends on the specific antibody used. Refer to manufacturer&#39;s information to obtain dilution criteria for a specific antibody. Multiple antibodies can be used together but must be raised against different species.</li>
	<li>Wash the sections three times in PBS for 15 min each.</li>
	<li>Incubate the sections overnight in secondary antibody (1:200, 1% BSA in PBS) at 4 &#176;C on a shaker. 
	NOTE: Multiple secondary antibodies can be used at different spectra if multiple primary antibodies were used.</li>
	<li>Wash off the secondary antibody by washing the sections three times in PBS for 10 min each.</li>
	<li>Mount and dry the sections on a glass slide for microscopy. 
	NOTE: The sections should still be moist prior to mounting the coverslip.</li>
	<li>Apply mounting medium suited for fluorescence dyes on the tissue sections and apply a coverslip.</li>
	<li>Dry the sections for 1 h.</li>
	<li>For long term storage, seal the coverslip using nail polish and keep in the dark at 4 &#176;C.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+trajectories+between+mouse+somatosensory+thalamus+and+cortex.">Axonal trajectories between mouse somatosensory thalamus and cortex.</a> J Comp Neurol. 258 (4), 542-564 (1987).</li><li>Ray, S., Burgalossi, A., Brecht, M., Naumann, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+Modular+Microcircuits+of+the+Rat+Medial+Entorhinal+Cortex.">Complementary Modular Microcircuits of the Rat Medial Entorhinal Cortex.</a> Front Syst Neurosci. 11, (2017).</li><li>Livingstone, M. S., Hubel, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamic+inputs+to+cytochrome+oxidase-rich+regions+in+monkey+visual+cortex.">Thalamic inputs to cytochrome oxidase-rich regions in monkey visual cortex.</a> Proc Natl Acad Sci U S A. 79 (19), 6098-6101 (1982).</li><li>Land, P. W., Simons, 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=Cytochrome+oxidase+staining+in+the+rat+SmI+barrel+cortex.">Cytochrome oxidase staining in the rat SmI barrel cortex.</a> J Comp Neurol. 238 (2), 225-235 (1985).</li><li>Welker, C., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+layer+IV+in+the+somatosensory+neocortex+of+the+rat:+description+and+comparison+with+the+mouse.">Structure of layer IV in the somatosensory neocortex of the rat: description and comparison with the mouse.</a> J Comp Neurol. 158 (4), 437-453 (1974).</li><li>Retzius, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+Cajal'schen+zellen+der+grosshirnrinde+beim+menschen+und+bei+s&#228;ugetieren.">Die Cajal'schen zellen der grosshirnrinde beim menschen und bei s&#228;ugetieren.</a> Biol Unters. 5, 1-9 (1893).</li><li>Cajal, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histologie du Systeme Nerveux de l'Homme et des vert&#233;br&#233;s. , (1911).</li><li>Chapin, J. K., Lin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+body+representation+in+the+SI+cortex+of+anesthetized+and+awake+rats.">Mapping the body representation in the SI cortex of anesthetized and awake rats.</a> J Comp Neurol. 229 (2), 199-213 (1984).</li><li>L&#246;wel, S., Freeman, B., Singer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+organization+of+the+orientation+column+system+in+large+flat-mounts+of+the+cat+visual+cortex:+A+2-deoxyglucose+study.">Topographic organization of the orientation column system in large flat-mounts of the cat visual cortex: A 2-deoxyglucose study.</a> J Comp Neurol. 255 (3), 401-415 (1987).</li><li>Tang, Q., 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=Functional+architecture+of+the+rat+parasubiculum.">Functional architecture of the rat parasubiculum.</a> J Neurosci. 36 (7), 2289-2301 (2016).</li><li>Snyder, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Map projections--A working manual (Vol. 1395). , (1987).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nat Methods. 10 (6), 508-513 (2013).</li><li>Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iDISCO:+a+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159 (4), 896-910 (2014).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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 histochemistry and (ii) immunohistochemical staining.
Cytochrome oxidase staining has been one of the most popular methods to visualize cortical modules, and has been widely used in the primary sensory cortices27,28. It visualizes modules by staining darker for areas of higher mitochondrial activity17, thereby acting as a proxy for anatomical modules which elicit substantial functional responses. This method has been used to visualize somatosensory cortical modules (Figure 2) and cortical modules in the visual cortex27.
One of the drawbacks of traditional histochemical techniques is that their typical visualization by bright-field light microscopy limits the number of modules that can be visualized simultaneously. However, immunohistochemical methods, in conjugation with fluorescent visualization probes can also be utilized to view particular proteins and identify cortical modules. For instance, thalamic afferents to the sensory cortices project in a somatotopic manner. Thus, visualizing these afferents, using VGluT2 immunoreactivity can also be used to visualize the body maps in the primary somatosensory cortex which we have visualized using cytochrome oxidase activity (Figure 2). Selective proteins can also be used as cellular markers to identify groups of genetically defined cells. Using immunohistochemistry, we identified clusters of pyramidal cells in the medial entorhinal cortex expressing the calcium binding protein, calbindin6 (Figure 3) and determined that clusters of this particular genetically determined group of cells are conserved across evolution5, indicating common microcircuit principles underlying their function. Using multiple fluorophores, we also delineated complementary modules in the medial entorhinal cortex26, demonstrating parallel microcircuits which underlie spatial memory.
Historically, the brain has been sectioned in a plane normal to its position in the body, in either sagittal, coronal, or horizontal orientations. The identification of cortical modules, like the barrel fields in the somatosensory cortex4 also prompted sectioning along a tangential and subsequently in flattened cortical preparations29, though isolated cases of such sectioning were observed much earlier30,31. Subsequent to the identification of the barrel cortex, other cortical modules were also identified in the primary visual cortex27, as well as the presence of a complete body map32 with a somatotopic representation of the body in primary somatosensory cortex. Flattened sections of the cat visual cortex also revealed the topographic organization of orientation columns without the need for additional reconstructions33. Cortical modularity has also been demonstrated in parahippocampal cortices, including the presubiculum and parasubiculum26,34 and medial entorhinal cortex6. Typically flattening a curved object induces distortions in laminar topography - as can be observed by the multitude of projections of the curved earth surface on a flat map35. A relatively simplistic method to partially compensate for these distortions is by introducing reference points at defined distances before flattening, and then measuring the distance between them after flattening. These marks then not only subserve in quantitatively estimating distortions introduced by flattening, but also provide an easy reference point to align consecutive sections. Further crucial aspects for preserving laminar fidelity include ensuring homogenous flattening across the section, and finally sectioning exactly tangential to the laminar layout to obtain optimized laminar cortical sections.
The visualization of cortical modules in flattened tangential sections has played an important part in revealing fascinating structure-function relationships in the cerebral cortex. The topographic representation of whisker and body parts in the primary somatosensory cortex and the presence of an anatomical grid in the microcircuits generating functional grid cells in flattened tangential cortical sections reveal intricate microcircuits details which would be hard to comprehend from other orientations. However, our current techniques provide only a two-dimensional slice of these essentially three-dimensional modules. Using immunofluorescence techniques and tissue clearing methods36,37, it would be possible to visualize these cortical modules as a whole and perhaps reveal further insights into our understanding of cortical maps and modules.

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>&lt;strong&gt;Cytochrome oxidase staining&lt;/strong&gt;</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&#039;Diaminobenzidine tetrahydrochloride hydrate</td><td>Sigma-Aldrich</td><td>D5637</td><td></td></tr><tr><td>D(+)-Saccharose</td><td>Carl Roth&amp;nbsp;</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&amp;nbsp;</td><td>9105.4</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antigen retrieval&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Phosphate buffer/phosphate-buffered saline/prefix/PFA&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunohistochemistry&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mounting or freezing media&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Alcohol dehydration&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Devices/tools&lt;/strong&gt;</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><td></td><td></td></tr><tr><td>Dumont #55 Forceps - Inox</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Dumont #5 Forceps - Inox Biology Tip</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>Dumont #5SF Forceps - Inox Super Fine Tip</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Bone Shears - 24 cm</td><td>Fine Science Tools</td><td>16150-24</td><td></td></tr><tr><td>Friedman Rongeur</td><td>Fine Science Tools</td><td>16000-14</td><td></td></tr><tr><td>Blunt Scissors</td><td>Fine Science Tools</td><td>14000-18</td><td></td></tr><tr><td>Surgical Scissors - Large Loops</td><td>Fine Science Tools</td><td>14101-14</td><td></td></tr><tr><td>Surgical Scissors - Sharp-Blunt</td><td>Fine Science Tools</td><td>14001-13</td><td></td></tr><tr><td>Fine Iris Scissors</td><td>Fine Science Tools</td><td>14094-11</td><td></td></tr></tbody></table>

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 contrast, other body parts such as paws and genitals show variations in their relative size and reflect the specialization to an ecological niche or to sexual selection22,23.
To understand the architecture of the medial entorhinal cortex, we obtained sections parallel to the pial surface. This was mainly achieved by tangential sectioning of the medial entorhinal cortex in mice, rats, and Egyptian fruit bats. In humans, because of the considerably larger size and more undulations in the entorhinal cortex, we gently flattened the cortex subsequent to making a tangential cut of the entorhinal cortex. Subsequently, all brains were cryopreserved and sectioned on a cryostat at 60 &#181;m. Immunohistochemistry was performed on the obtained sections with an anti-calbindin antibody, to visualize the calbindin-positive pyramidal cell modules in the medial entorhinal cortex5 (Figure 3). The calbindin-modules in the entorhinal cortex show a remarkable periodicity across all these brains, and vary in size by only a factor of 10 across ~ 20,000-fold variation in brain sizes5.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56992/56992fig2.jpg" /> 
Figure 2: Topographical layout of the barrel cortex modules across mammals identified by cytochrome oxidase histochemistry. Tangential sections of layer IV from the somatosensory cortex of (A) mouse, (B) Mongolian gerbil, (C) rat, (D) degu, (E) hamster, (F) rabbit, showing barrels as a highly conserved somatotopic representation of the mystacial vibrissae. Scale bars = 500 &#181;m. M: Medial, L: Lateral, R: Rostral, C: Caudal; Orientation in A also applies to B-E. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56992/56992fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56992/56992fig3.jpg" /> 
Figure 3: Periodic layout of medial entorhinal cortex modules across mammals identified by calbindin immunoreactivity. Tangential sections from layer II of the medial entorhinal cortex of (A) mouse, (B) rat, (C) Egyptian fruit bat, and (D) human, showing a conserved periodic layout of calbindin-positive pyramidal cell modules. Scale bars = 250 &#181;m. M: Medial, L: Lateral, D: Dorsal, V: Ventral, R: Rostral, C: Caudal; Orientation in D also applies to B, C. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56992/56992fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

visualization-of-cortical-modules-in-flattened-mammalian-cortices

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.7K Views.  Humboldt University of Berlin. This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Video: Visualization of Cortical Modules in Flattened Mammalian Cortices

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Medicine

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Behavior

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Biology

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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 ...

Preparation of Colliculo-Thalamocortical Slices from a Mouse Pup Brain

Source: Slater, B. J., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53067">Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging.</a> J. Vis. Exp. (2015)
The video demonstrates the preparation of colliculo-thalamocortical brain slices from mouse pup brains. An isolated brain is aligned on a marked slide and cut at an angle to align the auditory midbrain and forebrain in the same plane. The brain is then sliced using a vibratome, and the slices containing the major auditory midbrain and forebrain structures are transferred to a holding chamber for preservation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation for and Removal of ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

Summary

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Chapters in this video

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Multi-layer Cortical Ca²⁺ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In vivo Imaging of Deep Cortical Layers using a Microprism

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

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

Preparation of Colliculo-Thalamocortical Slices from a Mouse Pup Brain