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.
Långa fallande fibrerna till ryggmärgen är väsentliga för transport, smärtupplevelse, och andra beteenden. Fiber avslutande mönster i ryggmärgen hos majoriteten av dessa fibersystem har inte undersökts grundligt i alla arter. Serotonerga fibrer, som skjuter ut till ryggmärgen, har studerats hos råttor och opossum på histologiska sektioner och deras funktionella betydelse har härletts baserat på deras fiber avslutande mönster i ryggmärgen. Med utvecklingen av CLARITY och kubiska tekniker, är det möjligt att undersöka denna fiber och dess fördelning i ryggmärgen, vilket sannolikt kommer att avslöja tidigare okända egenskaper hos serotonerga supraspinala vägar. Här ger vi ett detaljerat protokoll för att avbilda de serotonerga fibrerna i ryggmärgen hos möss med användning av kombinerade CLARITY och kubiska tekniker. Metoden innebär perfusion av en mus med en hydrogel lösning och förtydligande av vävnaden med en combination att rensa reagens. Ryggmärgsvävnad rensades i knappt två veckor, och den efterföljande immunofluorescerande färgning mot serotonin fördes på mindre än tio dagar. Med en multi-foton fluorescerande mikroskop, var vävnaden scannas och en 3D-bild rekonstruerades med hjälp av Osirix programvara.
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.
Det protokoll som beskrivits visar hur bilden serotonerga fibrer i ryggmärgen hos möss med den kombinerade CLARITY och kubiska tekniker. Det införs en snabbare clearing process jämfört med den passiva clearing protokoll som utvecklats av et al. Cheung 14 och al. Tomer et 15 och tillåter ryggmärg vara väl stöds av hydrogelen under clearing.
Ett viktigt steg under fixering av ryggmärgen hos möss, som rapporterats av et al. Ch…
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/ |