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Neuroscience

CLARITY / CUBIC技術を用いたマウスの脊髄にセロトニン繊維のイメージング

Published: February 26, 2016
doi:
10.3791/53673
, ,
1Brain Structure and Function Group,Neuroscience Research Australia, 2School of Medical Sciences,The University of New South Wales

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

脊髄への長い下降繊維が移動、疼痛知覚、およびその他の動作のために不可欠です。これらのファイバシステムの大部分の脊髄における繊維の終端パターンを徹底的に任意の種に調べられていません。脊髄に突出セロトニン作動性線維は、組織切片のラットおよびオポッサムに研究されており、それらの機能的意義は、脊髄でのファイバ終端パターンに推定基づいています。 CLARITY立方技術の発展に伴い、このファイバシステムとセロトニン脊柱上の経路の未知の機能を明らかにする可能性がある脊髄におけるその分布を調べることが可能です。ここでは、合成CLARITY立方技術を使用して、マウスの脊髄におけるセロトニン線維を撮像するための詳細なプロトコルを提供します。この方法は、COMBINと組織のヒドロゲル溶液と明確化とマウスの灌流を伴います試薬をクリアするエーション。脊髄組織は、すぐ下に2週間でクリアされた、およびセロトニンに対するその後の免疫染色は、10日以内に完了しました。多光子蛍光顕微鏡を用いて、組織を走査し、3次元画像は、OsiriXのソフトウェアを使用して再構築しました。

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

倫理文：動物を対象とするすべての手順は、ニューサウスウェールズ大学の動物管理及び倫理委員会（ACEC）（承認ACEC番号は14 / 94Aである）のガイドラインに従ってください。 透明なマウス脊髄の調製アイスコールドヒドロゲル溶液の調製 16％パラホルムアルデヒド溶液（PFA）の調製 70ミリリットル予め温めておいた蒸留水（50〜55℃）でに16グラムの?…

Representative Results

このセクションでは、CLARITY立方プロトコルの組み合わせを使用して、透明なマウスの脊髄におけるセロトニン抗体染色の結果を示します。私たちは、セロトニン作動性繊維は（もビデオ1参照、 図1）前角の腹の部分で優勢で脊髄の全ての薄層に存在していることを示しています。対照組織は陽性線維（結果は表示されませんでした）を持っ?…

Discussion

プロトコルを組み合わせCLARITYとCUBIC技術を用いてマウスの脊髄における画像セロトニン作動性繊維にどのようにショーを説明しました。これは、チャンら 14およびさまら 15によって開発された受動的な決済プロトコルに比べて速くクリア処理を紹介し、脊髄組織が ​​うまくクリア時にヒドロゲルによってサポートすることができます。

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Disclosures

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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References

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Tags

CLARITY/CUBIC TechniqueSerotonergic FibersMouse Spinal CordNeuroanatomyTraditional Histology3D FeaturesIn Situ ObservationVectorsCentral Nervous SystemCardiovascular SystemTracing MethodPlastic Dissection StageFume HoodAnesthetized MouseChest OpeningHeart ExposurePeristaltic Pump TubingSaline WashBlood Perfusion
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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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