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 magn...

<ol>
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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.

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			<td height="22" style="height:22px;width:495px;">Photoinitiator VA044</td>
			<td style="width:495px;">Wako</td>
			<td style="width:284px;">va-044/225-02111</td>
			<td style="width:807px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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>
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		<tr height="21">
			<td height="21" style="height:21px;">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=5e7a4f31-879c-4d63-ba0b-82556a0ccf1d</td>
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			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td align="right">158127</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/158127?lang=en&#38;region=AU</td>
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			<td height="21" style="height:21px;">urea</td>
			<td>Merck Millipore</td>
			<td align="right">66612</td>
			<td>http://www.merckmillipore.com/AU/en/product/Urea---CAS-57-13-6---Calbiochem,EMD_BIO-66612</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis (2-hydroxypropyl) ethylenediamine</td>
			<td>Merck Millipore</td>
			<td align="right">821940</td>
			<td>http://www.merckmillipore.com/AU/en/product/Ethylenediamine-N,N,N&#39;,N&#39;-tetra-2-propanol,MDA_CHEM-821940</td>
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			<td height="21" style="height:21px;">Triton-X 100</td>
			<td>Merck Millipore</td>
			<td align="right">648462</td>
			<td>http://www.merckmillipore.com/AU/en/product/TRITON&#174;-X-100-Detergent---CAS-9002-93-1---Calbiochem,EMD_BIO-648462</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s0389?lang=en&#38;region=AU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8217;,2&#8217;&#8217;- nitrilotriethanol</td>
			<td>Merck Millipore</td>
			<td align="right">137002</td>
			<td>http://www.merckmillipore.com/AU/en/product/Triethanolamine-(Trolamine),MDA_CHEM-137022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">goat anti rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 594 conjugate</td>
			<td>Life Technologies</td>
			<td>&#160;A-11012</td>
			<td>https://www.lifetechnologies.com/order/genome-database/antibody/Rabbit-IgG-H-L-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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>
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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...

Watch this Scientific Journal Video about Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique at JoVE.com

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

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 exit point for the blood.Then, wash the heart with 40 milliliters of 9 percent saline at approximately 10 milliliters per minute. When all of the blood has been flushed, perfuse the animal with 35 milliliters of freshly prepared, ice cold hydrogel at the same perfusion rate. Then, incise the skin over the back of the mouse and dissect the muscles nest to the vertebrae.After cutting the vertebral arches on one side and flipping them to the other, excise the spinal cord from the vertebral column with fine scissors. Immediately after harvesting, place the spinal cord in 15 milliliters of freshly prepared hydrogel solution overnight at four degrees Celsius. The next day, remove 10 milliliters of the hydrogel and pour the rest of the solution and spinal cord into a five milliliter tube.Add fresh hydrogel solution until the tube is full and then stretch a small piece of parafilm over the top of the tube, taking care that the parafilm contacts the hyrdogel and that there are no bubbles between the solution and the film. Removing air from the tube is the most difficult step. To make sure there are no bubbles, take care to fill the tube slowly with the hydrogel solution and then carefully place the parafilm across the top of the tube, without spilling the gel.Now, wrap a second piece of parafilm around the neck of the tube and incubate the spinal cord in a 37 degree Celsius oven overnight. When the hydrogel solution becomes a gel, use a spanner to remove the gel and neural tissue from the tube. To remove the hydrogel from the spinal cord, briefly press a course tissue against the gel.The gel will stick to the tissue as it is removed. When the spinal cord is clean, place it in PBS for four, six hour washes on a shaker. After 24 hours, use a razor blade to cut the spinal cord coronally into two to three millimeter long segments and transfer the tissue pieces into five milliliters of freshly prepared CUBIC clearing solution.Shake the pieces in the 37 degree Celsius oven for 72 hours and then replace the three day old clearing solution with freshly prepared clearing solution. Two to three days later, check the transparency of the tissue against the paper with font size 8 letters. When the letters can be observed through the cleared tissue, replace the CUBIC clearing solution with four milliliters of fresh PBST for four, six hour washes.To stain the spinal cord segments for fluorescent imaging, incubate the tissue in the appropriate primary antibody solution for three days on a shaker in the 37 degree Celsius oven. At the end of the primary antibody staining, immerse the tissue in four milliliters of PBST for four, six hour washes in the 37 degree Celsius oven. Then, replace the PBST with the appropriate secondary antibody solution for three more days on the shaker in the 37 degree Celsius oven.On the fourth day, wash away the unbound secondary antibody with PBST, as just demonstrated. To image the segments, replace the last PBST wash with four milliliters of 85 percent glycerol to make the refractive index of the tissue even. After about an hour, check the transparency of the tissue.When the tissue is clear, transfer the spinal cord segments on to a large, thin glass slide. Next, place a few drops of 85 percent glycerol next to the spinal cord tissue and cover the tissue with a 22 by 50 millimeter glass cover slip. Place the slide into the holding frame of the microscope and move the tissue into the light path.Then, select the Helium Neon laser and check the brightness of the positive signal in the live image to adjust the intensity of the laser. With the laser at the optimal intensity, use a 20x objective to select the scanning area of the spinal cord tissue. Then, set the instrument to create a z-stack with the depth of each stack at three microns.Finally, scan the tissue from the top to the bottom of the z-stack, first, under the 20x objective, followed by a z-stack scan under the 63x objective. 3D software can then be used to reconstruct the appropriate 3D videos. Here, the presence of serotonergic fibers in all of the laminae of the spinal cord, with a predominance in the ventral portion of the ventral horn, can be observed.In the ventral horn, densely packed serotonergic fibers are present in the ventral medial area, extending toward the lateral portion of the ventral horn, with some of the immunopositive fibers also extending from the ventral horn, toward the dorsal horn, or central canal. Immunopositive fibers are present in all of the laminae of the dorsal horn, but there is a small gap between the positive fibers in laminae two and four, particularly, in the lateral part of the dorsal horn. In a horizontal section, densely packed sertonergic fibers in ventral horn along the longitudinal axis of the spinal cord, can be observed issuing branches regularly along the axis perpendicular to the fiber bundle.These branches further diverge along their paths, to the lateral portion of the ventral horn. Compared with these branches, those extending toward the midline are more irregular and smaller. Under the 63x objective, the serotonergic fibers in the dorsal horn, are observed to travel along the axis of the spinal cord and to terminate at various points along the path of the tract with thick fibers, sporadically distributed among the thin fibers.Once mastered, this technique can be completed in less than three weeks, if it is performed properly. Following this procedure, other methods, like double labeling with choline acetyltransferase antibody, can be performed to answer additional questions about whether serotonergic fibers terminate motor neurons. After its development, this technique paved the way for researchers in the field of biomedical imaging to explore the complex spatial relationships between various cell types in animal specimens or human tissues.After watching this video, you should have a good understanding of how to clarify the mouse spinal cord tissue for 3D imaging after immunofluorescent staining.

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Research

JoVE Journal

Neuroscience

10.4K vistas.  Neuroscience Research Australia. 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 traditio...