Summary

Визуализация Серотонинергической волокон в спинном мозге мышей Использование Четкость / КУБИЧЕСКИЙ Техника

Published: February 26, 2016
doi:

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

Длинные нисходящих волокон к спинному мозгу необходимы для передвижения, восприятия боли, а также другие виды поведения. Схема заделки оптоволокна в спинном мозге большинства этих волоконных систем не были тщательно исследованы в любых видов. Серотонинергической волокна, которые выступают в спинной мозг, были изучены у крыс и опоссумов на гистологических срезов и их функциональное значение было выведено на основе их рисунка заделки оптоволокна в спинном мозге. С развитием четкостью и КУБИЧЕСКИХ методов, можно исследовать эту систему волокна и его распределение в спинном мозге, который, вероятно, выявить ранее неизвестные особенности серотонинергической супраспинальных путей. Здесь мы приводим подробный протокол для визуализации серотонинергической волокон в спинном мозге мыши с использованием комбинированного ЯСНОСТИ и кубические техники. Способ включает перфузию мыши с раствором гидрогеля и осветления ткани с Комбенвания очистки реагентов. Ткани спинного мозга был очищен в чуть менее двух недель, и последующее окрашивание иммунофлуоресцентного против серотонина было завершено менее чем за десять дней. С многофотонном флуоресцентного микроскопа, ткань была отсканирована и 3D-изображение было реконструировано с помощью программного обеспечения 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

Этика Заявление: Все процедуры, связанные с предметов животного происхождения следуйте рекомендациям по уходу и комитетом по этике животных (ACEC) в Университете Нового Южного Уэльса (утвержденный номер ACEC на 14 / 94А). 1. Приготовление прозрачного спинного мозга мыши П…

Representative Results

В этом разделе показаны результаты окрашивания серотонин антител в прозрачной мыши спинного мозга с использованием комбинации Четкость и кубические протоколов. Показано , что серотонинергические волокна присутствуют во всех пластинках спинного мозга с преобладани…

Discussion

Протокол, описанный показывает, как изображение серотонинергических волокон в спинном мозге мыши с комбинированным четкостью и КУБИЧЕСКИХ техники. Он вводит более быстрый процесс клиринга по сравнению с пассивным протоколом клиринга , разработанного Cheung и др. 14 и Томер и

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&region=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&region=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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Citar este artículo
Liang, H., Schofield, E., Paxinos, G. Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique. J. Vis. Exp. (108), e53673, doi:10.3791/53673 (2016).

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