Zheng Huiyuan, Rinaman Linda
Department of Neuroscience, University of Pittsburgh, A210 Langley Hall, Pittsburgh, PA, 15260, USA.
Brain Struct Funct. 2016 May;221(4):2375-83. doi: 10.1007/s00429-015-1020-0. Epub 2015 Mar 14.
CLARITY is an innovative technological advance in which intact biological tissue is transformed into a "nanoporous hydrogel-hybridized form" (Chung et al. 2013; Chung and Deisseroth 2013) with markedly improved chemical and optical accessibility, permitting fluorescent visualization and extraction of high-resolution structural data from mm-thick blocks of tissue. CLARITY affords an excellent but as yet unexploited opportunity to visualize the growth and maturation of phenotypically identified neurons and axonal processes in the developing brain. This brief report describes a moderately revised, simplified, and less expensive CLARITY protocol that effectively reveals the structure of chemically identified neurons in whole neonatal/juvenile rat brains and tissue slabs. Rats [postnatal day (P)0-24] were transcardially perfused with one of two fixative/hydrogel solutions, followed by hydrogel polymerization to generate brain hybrids. Whole brain hybrids or 2.0-mm-thick coronal slabs were passively cleared of lipid and then processed for dual immunofluorescence labeling, including labeling using tyramide signal amplification. After refractive index matching using 2,20-Thiodiethanol (60 % solution), a Leica confocal microscope equipped with a CLARITY objective was used to view the hypothalamus in whole brain hybrids or slabs. Collected image stacks revealed the distribution and three-dimensional structure of hypothalamic pro-oxyphysin (oxytocin)-, neuropeptide Y-, glucagon-like peptide-1-, and tyrosine hydroxylase-immunopositive neurons and processes within large tissue volumes. Outstanding structural preservation and immunolabeling quality demonstrates the efficacy of this approach for interrogating chemically defined neural circuits as they develop in postnatal rodent brain.
CLARITY是一项创新性的技术进展,通过该技术,完整的生物组织被转化为“纳米多孔水凝胶杂交形式”(Chung等人,2013年;Chung和Deisseroth,2013年),其化学和光学可及性显著提高,从而能够从毫米厚的组织块中进行荧光可视化并提取高分辨率结构数据。CLARITY为可视化发育中大脑中表型鉴定的神经元和轴突的生长与成熟提供了一个绝佳但尚未得到充分利用的机会。本简要报告描述了一种经过适度修订、简化且成本较低的CLARITY方案,该方案能有效揭示新生/幼年大鼠全脑及组织切片中化学鉴定神经元的结构。对出生后0至24天的大鼠进行经心灌注,使用两种固定剂/水凝胶溶液之一,随后进行水凝胶聚合以生成脑杂交体。对全脑杂交体或2.0毫米厚的冠状切片进行脂质被动清除,然后进行双重免疫荧光标记,包括使用酪胺信号放大进行标记。使用2,20 - 硫代二乙醇(60%溶液)进行折射率匹配后,使用配备CLARITY物镜的徕卡共聚焦显微镜观察全脑杂交体或切片中的下丘脑。收集的图像堆栈揭示了下丘脑促催产素(催产素)、神经肽Y、胰高血糖素样肽 - 1和酪氨酸羟化酶免疫阳性神经元及其突起在大体积组织中的分布和三维结构。出色的结构保存和免疫标记质量证明了该方法在研究出生后啮齿动物大脑中发育的化学定义神经回路方面的有效性。