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.
fibres descendantes longues à la moelle épinière sont essentiels pour la locomotion, la perception de la douleur, et d'autres comportements. Le motif de terminaison des fibres dans la moelle épinière de la plupart de ces systèmes de fibres n'a pas été complètement étudiée chez toutes les espèces. fibres sérotoninergiques, qui projettent de la moelle épinière, ont été étudiés chez des rats et des opossums sur des coupes histologiques et leur signification fonctionnelle a été déduite en fonction de leur motif de terminaison de fibre dans la moelle épinière. Avec le développement de CLARITY et techniques CUBES, il est possible d'étudier ce système de fibre et de sa distribution dans la moelle épinière, ce qui est susceptible de caractéristiques inconnues des voies sérotoninergiques supraspinales révéler. Ici, nous fournissons un protocole détaillé pour l'imagerie des fibres sérotoninergiques dans la moelle épinière de souris à l'aide du CLARITY combiné et techniques CUBES. Le procédé implique la perfusion d'une souris avec une solution d'hydrogel et la clarification du tissu avec une Combination de la compensation des réactifs. le tissu de la moelle épinière a été effacé en un peu moins de deux semaines, et la coloration immunofluorescence ultérieure contre la sérotonine a été achevée en moins de dix jours. Avec un microscope à fluorescence multi-photon, le tissu a été numérisé et une image en 3D a été reconstruit en utilisant un logiciel 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.
Le protocole décrit montre comment l'image des fibres sérotoninergiques dans la moelle épinière de souris avec le CLARITY combinés et techniques CUBES. Elle introduit un processus de compensation plus rapide par rapport au protocole de compensation passive développée par Cheung et al. 14 et Tomer et al. 15 et permet le tissu de la moelle épinière à être bien pris en charge par l'hydrogel lors de la compensation.
Une étape importante…
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/ |