Name: Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique (Video) | JoVE Journal
Uploaded: 2016-02-26T14:00:00.000+00:00
Duration: 9 min 54 s
Description: Learn the CLARITY/CUBIC technique to visualize serotonergic fibers in the mouse spinal cord in 3D. This method reveals fiber distributions and vectors invisible to traditional histology for neuroanatomy research.

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This work was supported by the Australian Research Council Centre of Excellence for Integrative Brain Function (ARC Centre Grant CE140100007), an NHMRC project grant (#1086643). Prof. George Paxinos is supported by a Senior Principal Research Fellow NHMRC grant (#1043626).

Ethics Statement: All procedures involving animal subjects follow the guidelines of the Animal Care and Ethics Committee (ACEC) at The University of New South Wales (the approved ACEC number is 14/94A).
1. Preparation of the Transparent Mouse Spinal Cord
<ol>
	<li>Preparation of Ice Cold Hydrogel Solution
	<ol>
		<li>Preparation of 16% paraformaldehyde solution (PFA)
		<ol>
			<li>Add 16 g paraformaldehyde powder into 70 ml pre-warmed distilled water (50-55 &#176;C) and stir on a heated magnetic stirrer until paraformaldehyde is dissolved. Note: Do not allow the solution to heat over 55&#160;&#176;C and be aware that paraformaldehyde is toxic.</li>
			<li>Transfer the paraformaldehyde solution to a cylinder and add distilled water to 100 ml. Cool the paraformaldehyde solution using a 4&#160;&#176;C refrigerator.</li>
		</ol>
		</li>
		<li>Dissolve 125 mg VA-044 initiator in 26.25 ml distilled water and cool the solution in a 4&#160;&#176;C fridge.</li>
		<li>Cool 5 ml acrylamide solution (40%), 1.25 ml Bis solution (2%) (CAUTION: Bis and acrylamide solutions are toxic, may cause genetic defects or cancer), 5 ml 10x PBS, 12.5 ml 16% PFA solution, and the VA-044 initiator solution on ice.</li>
		<li>Mix the above solutions on ice. 
		Note: the total volume is 50 ml.</li>
	</ol>
	</li>
	<li>Perfusion and Collection of the Mouse Spinal Cord
	<ol>
		<li>Anaesthetize a mouse with an intraperitoneal injection of ketamine (80 mg/kg) and xyzaline (5 mg/kg) diluted in 0.9% normal saline.&#160;&#160;The dosage for mouse can be lower than that for rats.</li>
		<li>On a suitable plastic surface in a fume hood, fix the limbs of the mouse away from the body (sticky tape is effective) once the mouse does not respond to a pinch to its skin of the foot.</li>
		<li>Cut the mouse skin over the chest and then the bone chest to expose the heart with a scissor.</li>
		<li>Using a peristaltic pump, with a 25 G needle attached to the end of the tubing, insert the needle into the left ventricle of the heart, snip the right atrium to create an exit point for the blood and then start washing with 40 ml 0.9% normal saline a rate of approximately 10 ml/min.</li>
		<li>When complete, perfuse the mouse with 35 ml ice cold hydrogel solution. 
		Note: perfuse at a speed of 10 ml/min to avoid swelling of the nervous tissue.</li>
		<li>Incise the skin over the back with a blade and then remove the muscles next to the vertebrate. After cutting the vertebral arches on one side and flipping them to the other side, take the spinal cord out of the vertebral column with a fine scissor.</li>
	</ol>
	</li>
	<li>Clearing the Mouse Spinal Cord
	<ol>
		<li>Place the spinal cord in 15 ml of hydrogel solution overnight at 4 &#176;C.</li>
		<li>Remove 10 ml of the hydrogel solution and pour the rest of the solution with the spinal cord segments to a 5 ml tube. Fill the 5 ml tube with the hydrogel solution until it is completely full.</li>
		<li>Stretch a small piece of parafilm over the top of the 5 ml tube and then wrap parafilm around the neck of the tube. Note: Ensure the parafilm contacts the hydrogel solution and there are no bubbles between the hydrogel solution and the parafilm.</li>
		<li>Put the 5 ml tube in a 37&#160;&#176;C oven overnight. Observe the hydrogel solution till it becomes a gel.</li>
		<li>Take the 5 ml tube out of the oven and remove the gel from the tube with a spanner (such as a shoveled 1 ml pipette tip). Remove the gel from the spinal cord tissue by sticking the coarse tissue to the gel and then take the coarse tissue away (the gel will stick to the tissue and therefore will be removed by the tissue).</li>
		<li>After removing the gel from the spinal cord tissue, wash the spinal cord 4 times with 1x PBS (pH 7.4) for 24 hr on a shaker.</li>
		<li>Prepare the CUBIC clearing solution by dissolving 3.85 g urea and 3.85 g N,N,N&#39;,N&#39;-tetrakis (2-hydroxypropyl) ethylenediamine in 5.38 ml distilled water. 
		Note: A hot stirrer is used. Add 2.31 g polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 to the solution once it is clear and cools down to room temperature. 
		Note: 10 g of these three chemicals are for one mouse.</li>
		<li>Cut the mouse spinal cord coronally into 2-3 mm long segments with a razor blade and put them into 5 ml of the above CUBIC clearing solution. Place on a shaker in the oven at 37&#160;&#176;C for 3 days.</li>
		<li>Three days later, change the clearing solution to a fresh one. Note: the above solution is prepared prior to use.</li>
		<li>Two to three days later after refreshing the CUBIC clearing solution, check the transparency of the tissue against a paper with font 8 letters. If it is transparent, the letters can be seen through the cleared tissue. Remove the clearing solution and add 4 ml of PBST (0.1% Triton-X100) to wash the tissue 4 times a day (every 6 hours).</li>
	</ol>
	</li>
</ol>
2. Immunofluorescence Staining
<ol>
	<li>Incubate the spinal cord segments in the primary antibody solution (anti-serotonin, raised in rabbit, diluted 1:100 in PBST) for 3 days on a shaker in a 37&#160;&#176;C oven.</li>
	<li>Remove the antibody solution and add 4 ml of PBST (0.1% Triton-X100) to wash the spinal cord segments 4 times a day (every 6 hr) in a 37&#160;&#176;C oven.</li>
	<li>Remove the PBST solution and add the secondary antibody solution (594 conjugated goat anti-rabbit IgG, diluted 1:100 in PBST) to incubate the tissue for 3 days on a shaker in a 37&#160;&#176;C oven.</li>
	<li>Remove the secondary antibody solution and add 4 ml of PBST to wash the spinal cord segments 4 times a day (every 6 hours) on a shaker in a 37&#160;&#176;C oven. The next day the tissue is ready for imaging.</li>
</ol>
3. Imaging
<ol>
	<li>Remove the PBST solution and add 4 ml of 85% glycerol to make the refractive index of the tissue even.</li>
	<li>Check the clarity of the tissue. Once it is clear in approximately 1 hr, put the tissue onto a large, thin glass slide which fits the holding frame of the multi-photo fluorescent microscope.</li>
	<li>Put a few drops of 85% glycerol next to the spinal cord tissue and coverslip it with a 22 x 50 mm2 coverslip glass. 
	Note: The orientation of the spinal cord decides how the image looks like.</li>
	<li>Put the glass slide onto the holding frame of the multi-photon fluorescent microscope and move the tissue into the light pathway.</li>
	<li>Choose the Helium Neon laser line 594 nm and adjust the intensity of the laser to the optimal level by checking the brightness of the positive signal in the live image.</li>
	<li>Select the scanning area of the spinal cord tissue using the 20X objective (in water, NA 0.7) and prepare to create a z-stack, and the depth of each step set to 3 &#181;m.</li>
	<li>Scan the tissue from the top to the bottom of the z-stack under the 20X objective and then 63X objective (in oil, NA 1.4) (step size was 1 &#181;m), respectively. Reconstruct 3D videos using a 3D software28.</li>
</ol>

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<li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues%5BTitle%5D)+AND+%22Nature+Protoc%22%5BJournal%5D)">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nature Protoc. 9 (7), 1682-1697 (2014).</li>
<li>Susaki, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157 (3), 726-739 (2014).</li>
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<li>Lee, H., Park, J. H., Seo, I., Park, S. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+application+of+the+electrophoretic+tissue+clearing+technology%2C+CLARITY%2C+to+intact+solid+organs+including+brain%2C+pancreas%2C+liver%2C+kidney%2C+lung%2C+and+intestine%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine.</a> BMC Dev Biol. 14, 781(2015).</li>
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</ol>

The authors have nothing to disclose.

The protocol described shows how to image serotonergic fibers in the mouse spinal cord with the combined CLARITY and CUBIC techniques. It introduces a faster clearing process compared to the passive clearing protocol developed by Cheung et al.14 and Tomer et al.15 and allows the spinal cord tissue to be well supported by the hydrogel during clearing.
An important step during fixation of the mouse spinal cord, as reported by Cheung et al.14 ...

Supraspinal projections are responsible for the modulation of diverse behaviors such as pain perception. One of the projections carrying nociceptive information contains serotoninergic fibers, which originate from the hindbrain raphe and adjacent reticular nuclei1,2. Physiological and pharmacological studies have demonstrated an increased release of serotonin in the dorsal horn of the spinal cord after electrical stimulation of the raphe nuclei in the hindbrain3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn6-8, but also in the intermediate zone7,9,10, the ventral horn7,11, and even lamina 1012,13. There are no similar studies in the mouse. The present study aimed to map the termination pattern of serotonergic fibers arising from the hindbrain raphe nuclei and their adjacent reticular nuclei in the mouse spinal cord using the recently published CLARITY14 method and its modification - CUBIC15. 
Conventional fluorescence or peroxidase immunohistochemistry of the spinal cord clearly shows the distribution of serotonergic fibers in the gray matter of the spinal cord in 30-40 &#181;m thick cross-sections. However, this approach does not show the continuity of the serotonergic fiber tracts in the white matter and their collaterals in the gray matter. Although the 3D reconstruction of histological sections has advanced our knowledge of fiber tracts, it remains a challenge for histologists and anatomists to follow a single tract due to small distortions in the tissue caused by cutting. To circumvent this obstacle a number of researchers have developed various protocols for making the whole tissue structure transparent, and collecting an image of unaltered tissue in a single video file17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group14,15, as well as CUBIC, developed by Susaki et al16 are the most successful. Since the publication of the protocols, many researchers have started using these techniques to investigate various aspects of biological tissues, including, not only the brain22-25, but also the heart, kidneys, intestine, and the lungs26,27. 
By fixing the mouse spinal cord with the hydrogel solution (CLARITY) and clearing with the CUBIC reagents (which is a much faster method than that described by the original CLARITY protocol14,15), a spinal cord tissue block of 2-3 mm long was cleared within two weeks and immunofluorescence staining for serotonin completed in eight days. With just a combination of chemical agents, conventional immunohistochemistry can be used to create an image of individual fiber tracts in a 3D video file in approximately one month.

<table><tbody><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>40% acrylamide solution</td><td>Bio Rad</td><td>161-0140</td><td>http://www.bio-rad.com/en-au/sku/161-0140-40-acrylamide-solution</td></tr><tr><td>2% Bis Solution</td><td>Bio Rad</td><td>161-0142</td><td>http://www.bio-rad.com/en-au/sku/161-0142-2-bis-solution?parentCategoryGUID=5e7a4f31-&lt;br/&gt;879c-4d63-ba0b-82556a0ccf1d</td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>158127</td><td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&amp;amp;region=AU</td></tr><tr><td>urea</td><td>Merck Millipore</td><td>66612</td><td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#039;,N&#039;-tetra-2-propanol,MDA_CHEM-821940</td></tr><tr><td>Triton-X 100</td><td>Merck Millipore</td><td>648462</td><td>http://www.merckmillipore.com/AU/en/product/TRITON&amp;reg;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td></tr><tr><td>sucrose</td><td>Sigma</td><td>S0389</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&amp;amp;region=AU</td></tr><tr><td>serotonin antibody</td><td>Merck Millipore</td><td>AB938</td><td>http://www.merckmillipore.com/AU/en/product/Anti-Serotonin-Antibody,MM_NF-AB938</td></tr><tr><td>goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&amp;reg; 594 conjugate</td><td>Life Technologies</td><td>&amp;nbsp;A-11012</td><td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>multi-photon microscope</td><td>Leica</td><td>Leica TCS SP5 MP STED</td><td>http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp5-mp/</td></tr></tbody></table>

imaging serotonergic fibers in the mouse spinal cord using the clarity/cubic technique

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

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

This section shows results from serotonin antibody staining in the transparent mouse spinal cord using a combination of the CLARITY and CUBIC protocols. We show that serotonergic fibers are present in all laminae of the spinal cord with a predominance in the ventral portion of the ventral horn (Figure 1, also see Video 1). The control tissue did not have positive fibers (result was not shown). In the ventral horn, densely packed serotonergic fibers are pr...

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

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

The overall goal of this CLARITY/CUBIC experiment is to reveal the 3D features of serotonergic fibers in the mouse spinal cord, which are not observable by traditional histology. This method can help answer key questions in the neuroanatomy field, such as how are serotonergic fibers distributed throughout the spinal cord? The main advantages of this technique are that it allows serotonergic fibers to be observed in situ, along with identification of vectors that are not observable by traditional histology.Although this method can provide insight into the organization of the central nervous system, it can also be applied to other systems, such as the cardiovascular system. We first sought to use this method when we reached limitations using a traditional tracing method to analyze serotonergic fibers in the mouse spinal cord. Begin by fixing the limbs of an anesthetized mouse to a plastic dissection stage in a fume hood.Confirm a lack of response to toe pinch and use a pair of scissors to open the skin over the chest. Next, open the chest to expose the heart and insert a 25 gauge needle attached to a peristaltic pump tubing into the left ventricle. Snip the right atrium to create an

imaging-serotonergic-fibers-mouse-spinal-cord-using-claritycubic

Research

JoVE Journal

Neuroscience

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 Cleared Mouse Brain Expressing YFP

Source: Pekarek, B. T., et. al., Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing. J. Vis. Exp. (2021)In this video, we demonstrate the preparation, imaging, and analysis of a cleared mouse brain to examine dendritic arbors, spine morphology, and neuronal distribution using confocal microscopy and 3D reconstruction techniques.

Confocal Microscopy Imaging of Intact Dendritic Arbors and Spine Morphology in a Cleared Mouse Brain

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

This study presents a protocol for extracting and preparing cleared whole-brain and spinal cord samples with preserved fluorescent signals, enhancing experimental efficiency and data integrity to advance neuroscience research.

Extraction and Tissue Clearing Preparation of Mouse Brain-Spinal Cord Samples

Learn the CLARITY/CUBIC technique to visualize serotonergic fibers in the mouse spinal cord in 3D. This method reveals fiber distributions and vectors invisible to traditional histology for neuroanatomy research.

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

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