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

Summary

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.

Abstract

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.

Introduction

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 nuclei^1,2. 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 hindbrain^3-5. In the rat and opossum, serotonergic raphespinal fibers have dense terminals, not only in the dorsal horn^6-8, but also in the intermediate zone^7,9,10, the ventral horn^7,11, and even lamina 10^12,13. 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 µm 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 file^17-21. So far, the clear, lipid-exchanged, acrylamide-hybridized rigid, imaging/ immunostaining compatible, tissue hydrogel (CLARITY) technique, developed by Deisseroth's group^14,15, 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 brain^22-25, but also the heart, kidneys, intestine, and the lungs^26,27.

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 protocol^14,15), 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.

Protocol

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

1. Preparation of Ice Cold Hydrogel Solution
   1. Preparation of 16% paraformaldehyde solution (PFA)
      1. Add 16 g paraformaldehyde powder into 70 ml pre-warmed distilled water (50-55 °C) and stir on a heated magnetic stirrer until paraformaldehyde is dissolved. Note: Do not allow the solution to heat over 55 °C and be aware that paraformaldehyde is toxic.
      2. Transfer the paraformaldehyde solution to a cylinder and add distilled water to 100 ml. Cool the paraformaldehyde solution using a 4 °C refrigerator.
   2. Dissolve 125 mg VA-044 initiator in 26.25 ml distilled water and cool the solution in a 4 °C fridge.
   3. 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.
   4. Mix the above solutions on ice.
      Note: the total volume is 50 ml.
2. Perfusion and Collection of the Mouse Spinal Cord
   1. Anaesthetize a mouse with an intraperitoneal injection of ketamine (80 mg/kg) and xyzaline (5 mg/kg) diluted in 0.9% normal saline.  The dosage for mouse can be lower than that for rats.
   2. 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.
   3. Cut the mouse skin over the chest and then the bone chest to expose the heart with a scissor.
   4. 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.
   5. 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.
   6. 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.
3. Clearing the Mouse Spinal Cord
   1. Place the spinal cord in 15 ml of hydrogel solution overnight at 4 °C.
   2. 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.
   3. 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.
   4. Put the 5 ml tube in a 37 °C oven overnight. Observe the hydrogel solution till it becomes a gel.
   5. 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).
   6. 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.
   7. Prepare the CUBIC clearing solution by dissolving 3.85 g urea and 3.85 g N,N,N',N'-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.
   8. 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 °C for 3 days.
   9. Three days later, change the clearing solution to a fresh one. Note: the above solution is prepared prior to use.
   10. 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).

2. Immunofluorescence Staining

1. 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 °C oven.
2. 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 °C oven.
3. 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 °C oven.
4. 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 °C oven. The next day the tissue is ready for imaging.

3. Imaging

1. Remove the PBST solution and add 4 ml of 85% glycerol to make the refractive index of the tissue even.
2. 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.
3. 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.
4. Put the glass slide onto the holding frame of the multi-photon fluorescent microscope and move the tissue into the light pathway.
5. 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.
6. 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 µm.
7. 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 µm), respectively. Reconstruct 3D videos using a 3D software²⁸.

Results

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...

Discussion

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 ...}

Materials

List of materials used in this article

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