Imagiologia Fibras Serotoninérgicos na medula espinhal mouse usando o CLARIDADE / Técnica CUBIC
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
fibras descendentes longos para a medula espinhal são essenciais para a locomoção, a percepção da dor, e outros comportamentos. O padrão de terminação da fibra na medula espinal da maioria destes sistemas de fibra não foram cuidadosamente investigados em qualquer espécie. fibras serotoninérgicos, que se projectam para a espinal-medula, foram estudados em ratos e em secções histológicas marsupiais e o seu significado funcional foi deduzida com base no seu padrão de terminação da fibra na medula espinal. Com o desenvolvimento de clareza e técnicas cúbicos, é possível investigar este sistema de fibra e a sua distribuição na medula espinal, a qual é susceptível de revelar características anteriormente desconhecidos de vias supraespinhais serotoninérgicos. Aqui, nós fornecemos um protocolo detalhado para geração de imagens das fibras serotoninérgicos na medula espinhal de rato usando o CLAREZA combinados e técnicas cúbicos. O método envolve a perfusão de um rato com uma solução de hidrogel e clarificação do tecido com uma combinção de limpar reagentes. tecido da medula espinhal foi liberado em pouco menos de duas semanas, e a coloração de imunofluorescência subsequente contra a serotonina foi concluída em menos de dez dias. Com um microscópio de fluorescência multi-fotão, o tecido foi digitalizado e uma imagem 3D foi reconstruído usando software Osirix.
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 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.
Protocol
Representative Results
Discussion
O protocolo descrito mostra como fibras imagem serotoninérgicos na medula espinhal de rato com a clareza combinadas e técnicas cúbico. Ele apresenta um processo de limpeza mais rápido em comparação com o protocolo de compensação passiva desenvolvido por Cheung et al. 14 e Tomer et ai. 15 e permite que o tecido da medula espinal a ser bem suportado pelo hidrogel durante a limpeza.
Um passo importante durante a fixação da medula espinhal de rat…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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/ |
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