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.
Las fibras largas de descender a la médula espinal son esenciales para la locomoción, la percepción del dolor, y otros comportamientos. El patrón de terminación de fibras en la médula espinal de la mayoría de estos sistemas de fibra no se han investigado a fondo en cualquier especie. fibras serotoninérgicos, que se proyectan a la médula espinal, se han estudiado en ratas y zarigüeyas en secciones histológicas y su significado funcional se ha deducido en base a su patrón de terminación de fibras en la médula espinal. Con el desarrollo de la claridad y técnicas cúbico, es posible investigar este sistema de fibras y su distribución en la médula espinal, que es probable que revelar características hasta ahora desconocidas de las vías supraespinales serotoninérgicos. A continuación, ofrecemos un protocolo detallado para obtener imágenes de las fibras serotoninérgicos en la médula espinal de ratón usando la claridad combinado y técnicas cúbicos. El método consiste en la perfusión de un ratón con una solución de hidrogel y la clarificación del tejido con una combinación de la limpieza de los reactivos. el tejido de la médula espinal fue despejada en menos de dos semanas, y la posterior tinción de inmunofluorescencia contra la serotonina se completó en menos de diez días. Con un microscopio de fluorescencia multi-fotón, el tejido fue escaneada y una imagen 3D se reconstruyó usando software Osirix.
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 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 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.
El protocolo descrito se muestra cómo las fibras de imagen serotoninérgicos en la médula espinal de ratón con la claridad combinado y técnicas cúbico. Se introduce un proceso de compensación más rápido en comparación con el protocolo de compensación pasiva desarrollada por Cheung et al. 14 y Tomer et al. 15 y permite que el tejido de la médula espinal a estar bien apoyado por el hidrogel durante la limpieza.
Un paso importante durante la fi…
The authors have nothing to disclose.
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).
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 |
2,2’,2’’- nitrilotriethanol | Merck Millipore | 137002 | http://www.merckmillipore.com/AU/en/product/Triethanolamine-(Trolamine),MDA_CHEM-137022 |
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/ |