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Neurosciences

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

Published: February 11, 2021
doi:
10.3791/62126
,
1Anatomical Sciences and Neurobiology,University of Louisville

Summary

本文描述了整个真菌样、环形和上颚味蕾的组织制备、染色和分析方法，这些味蕾始终如一地产生完整和完整的味蕾（包括支配它们的神经纤维），并保持味蕾内结构与周围之间的关系。

Abstract

味蕾是味觉转换细胞的集合，专门用于检测口腔中化学刺激的子集。这些转导细胞与神经纤维进行通信，神经纤维将这些信息传递到大脑。由于味觉转换细胞在整个成年期不断死亡并被替换，因此味蕾环境既复杂又动态，需要对其细胞类型，位置以及它们之间的任何物理关系进行详细分析。详细的分析受到舌组织异质性和密度的限制，这些异质性和密度显著降低了抗体通透性。这些障碍需要切片方案，导致将味蕾分裂到各部分之间，以便测量值仅为近似值，并且丢失细胞关系。为了克服这些挑战，本文描述的方法涉及从三个味觉区域收集，成像和分析整个味蕾和单个末端乔木：真菌状，环状和上颚。收集整个味蕾可减少偏差和技术变异性，并可用于报告特征的绝对数字，包括味蕾体积，总味蕾神经支配，转导细胞计数和单个末端乔木的形态。为了证明这种方法的优点，本文使用一般的味蕾标记和所有味觉纤维的标签，对真菌样和环形味蕾之间的味蕾和神经支配体积进行了比较。还提供了使用味觉神经元稀疏细胞遗传标记（具有味觉转导细胞的标记子集）的工作流程。该工作流程使用图像分析软件分析单个味觉神经乔木的结构，细胞类型数以及细胞之间的物理关系。总之，这些工作流程为组织制备和分析整个味蕾及其支配乔木的完整形态提供了一种新颖的方法。

Introduction

味蕾是50-100个专门的上皮细胞的集合，它们结合口腔中存在的化学味觉刺激的亚群。味觉转换细胞通常被认为是1型、2型、3型、4型、5型、6型、7型、8型、9型，最初是基于电子显微镜标准，后来与分子标记相关。II型细胞表达磷脂酶C-β2（PLCβ2）2和瞬时受体电位阳离子通道，亚家族M成员51包括转导甜味、苦味和鲜味1、10的细胞。III型细胞表达碳酸酐酶4（Car4）11和突触体相关蛋白258，并表示主要对酸味11作出反应的细胞。转导咸度的细胞没有明确描绘12，13，14，但可能包括I型，II型和III型细胞15，16，17，18，19。味蕾环境是复杂和动态的，因为味觉转换细胞在整个成年期不断翻转，并被基底祖3，20，21取代。这些味觉转换细胞连接到来自膝状神经节和岩神经节的假单极神经纤维，后者将味觉信息传递到脑干。这些神经元主要根据它们携带的味觉信息22，23进行分类，因为直到最近24，关于它们形态的信息一直难以捉摸。II型细胞通过钙稳态调节蛋白1离子通道25与神经纤维通信，而III型细胞通过经典突触8，26进行通信。味蕾细胞的进一步表征 – 包括转导细胞类型谱系，影响其分化的因素以及连接乔木的结构都是积极研究的领域。

味蕾研究受到一些技术挑战的阻碍。构成舌头的异质性和致密组织显着降低了免疫组织化学的抗体通透性27，28，29。这些障碍使得切片方案成为必要，导致味蕾在各部分之间分裂，以便根据代表性部分近似测量或跨部分相加。以前，代表性薄片用于近似体积值和探头计数30。较厚的串行切片允许对所有味蕾切片进行成像，并对每个部分31的测量值进行求和。切割如此厚的切片并仅选择整个味蕾偏向于较小的味蕾32，33，34。来自切片味蕾的神经神经支配估计是基于对像素数13，35的分析，如果量化在所有36，37，38。这些测量完全忽略了单个神经心轴的结构和数量，因为心轴是分裂的（并且通常标记不良）。最后，虽然剥离上皮确实允许整个味蕾被染色39，40，但它也去除味蕾神经纤维，并可能破坏细胞之间的正常关系。因此，由于染色方法引起的这种破坏，对味蕾内结构关系的研究受到限制。

全结构收集消除了对代表性切片的需求，并允许确定体积，细胞计数和结构形态的绝对值测量41。这种方法还可以提高准确性，限制偏差并减少技术可变性。最后一个元素很重要，因为味蕾在34、42和跨区域43、44和整个味蕾分析中都显示出相当大的生物变异性，因此可以在对照和实验条件之间比较绝对细胞数量。此外，收集完整味蕾的能力允许分析不同转导细胞与其相关神经纤维之间的物理关系。因为变味细胞45可以相互交流，并且确实与神经纤维46通信，这些关系对正常功能很重要。因此，功能丧失条件可能不是由于细胞的丧失，而是由于细胞关系的变化。这里提供了一种收集整个味蕾的方法，以实现绝对测量的好处，以改进味蕾及其神经支配，味细胞计数和形状的体积分析，并促进转导细胞关系和神经乔木形态的分析。这种用于组织制备的新型全贴装方法的下游还介绍了两种工作流程：1）用于分析味蕾体积和总神经支配，2）用于味觉神经元的稀疏细胞遗传标记（标记了味觉转导细胞的子集），以及随后分析味神经乔木形态，味觉细胞类型的数量及其形状，以及使用图像分析软件来分析转导细胞与转导细胞之间的物理关系。 细胞及其神经乔木。总之，这些工作流程为组织制备以及整个味蕾及其支配乔木的完整形态的分析提供了一种新颖的方法。

Protocol

注意：所有动物都按照美国公共卫生服务政策关于实验动物的人道护理和使用以及NIH实验动物护理和使用指南制定的指南进行护理。Phox2b-Cre小鼠（MMRRC菌株034613-UCD，NP91Gsat / Mmcd）或TrkBCreER 小鼠（Ntrk2tm3.1（cre / ERT2）Ddg）与tdTomato报告小鼠（Ai14）一起繁殖。AdvillinCreER47 与Phox2b-flpo48 和Ai65一起繁殖。对于5-乙炔基-2′-脱氧尿苷（EdU）…

Representative Results

用dsRed和角蛋白-8（一般味蕾标记物）抗体染色舌上皮，标记整个味蕾和所有味蕾神经支配的Phox2b-Cre：tdTomato小鼠50，51（图3A）。对这些味蕾从毛孔到基底的成像给出了最高分辨率的x-y平面图像（图3A，B）。基于像素的成像程序的轮廓函数被用来勾勒出每个部分的味蕾外围（图3B）…

Discussion

开发一种持续收集和染色来自三个口腔味觉区域（真菌形，环状和味觉）的整个味蕾的方法，为分析味觉转导细胞，跟踪新掺入的细胞，神经支配以及这些结构之间的关系提供了显着的改进。此外，它有助于在标记群体50内或外定位潜在的二级神经元标记物。这一点尤其重要，因为味觉也接受强大的躯体感觉神经支配52 ， 53 ，这也可能标记一些味觉神经元。<sup…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢 Kavisca Kuruparanantha 对组织染色和环状味蕾成像的贡献， Jennifer Xu 对的神经支配进行染色和成像， Kaytee Horn 进行动物护理和基因分型，以及 Liqun Ma 对软腭味蕾的组织染色。该项目由R21 DC014857和R01 DC007176到R.F.K和F31 DC017660到L.O.支持。

Materials

2,2,2-Tribromoethanol ACROS Organics AC421430100
2-Methylbutane ACROS 126470025
AffiniPure Fab Fragment Donkey Anti-Rabbit IgG Jackson ImmunoResearch 711-007-003 15.5μL/mL
Alexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG Jackson Immuno Research 712-605-150 (1:500)
AutoQuant X3 software  Media Cybernetics
Blunt End Forceps Fine Science Tools  FST 91100-12
Click-iT™ Plus EdU Cell Proliferation Kit Molecular Probes C10637 Follow kit instructions 
Coverglass Marienfeld 107242
Cytokeratin-8 Developmental Studies Hybridoma Bank (DSHB), (RRID: AB_531826)  Troma1 supernatant (1:50, store at 4°C)
Dissection Scissors (coarse) Roboz RS-5619
Dissection Scissors (fine) Moria MC19B
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ThermoFisher Scientific A21206 (1:500)
Donkey anti-Rabbit, Alexa Fluor® 555 ThermoFisher Scientific A31572 (1:500)
DyLight™ 405 AffiniPure Fab Fragment Bovine Anti-Goat IgG Jackson Immuno Research 805-477-008 (1:500)
Fluoromount G Southern Biotech 0100-01
Glass slides Fisher Scientific (Superfrost Plus Miscroscope Slides) 12-550-15
Goat anti-Car4 R&D Systems  AF2414 (1:500)
Imaris  Bitplane  pixel-based image analysis software
Neurolucida 360 + Explorer MBF Biosciences 3D vector based image analysis software
Normal Donkey Serum Jackson Immuno Research 017-000-121
Normal Rabbit Serum  Equitech-Bio, Inc SR30
Olympus FV1000 (multi-Argon laser with wavelengths 458, 488, 515 and additional HeNe lasers emitting 543 and 633)
Paraformaldehyde EMD PX0055-3 4% in 0.1M PB
Rabbit anti-dsRed Living Colors DsRed Polyclonal Antibody; Clontech Clontech Laboratories, Inc. (632496) 632496 (1:500)
Rabbit anti-PLCβ2  Santa Cruz Biotechnology Cat# sc-206 (1:500)
Sodium Phosphate Dibasic Anhydrous Fisher Scientific BP332-500
Sodium Phosphate Monobasic Fisher Scientific BP330-500
tert-Amyl alcohol Aldrich Chemical Company 8.06193
Tissue Molds Electron Microscopy Sciences 70180
Tissue-Tek® O.C.T. Compound Sakura 4583
Triton X-100 BIO-RAD #161-0407
Zenon™ Alexa Fluor™ 555 Rabbit IgG Labeling Kit ThermoFisher Scientific Z25305 Follow kit instructions 
DOWNLOAD MATERIALS LIST

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Tags

Whole-mount StainingTaste BudsFungiformCircumvallatePalateLingual EpitheliumInnervationTransducing CellsOCT Compound

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Ohman, L. C., Krimm, R. F. Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds. J. Vis. Exp. (168), e62126, doi:10.3791/62126 (2021).
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