Bragg Elise M, Briggs Farran
Physiology & Neurobiology, Geisel School of Medicine at Dartmouth.
Physiology & Neurobiology, Geisel School of Medicine at Dartmouth;
J Vis Exp. 2017 Feb 15(120):55133. doi: 10.3791/55133.
This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population. The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing "Golgi-like" complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills. Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.
本方案概述了神经元的大规模重建，并结合使用独立且无偏的聚类分析，以全面调查在选择性神经元群体中观察到的形态特征。这些技术的结合构成了一种收集和分析神经解剖学数据的新方法。这些技术共同实现了对选择性神经元群体的大规模采样，从而更全面，并建立了无偏定量方法来描述群体中形态独特的神经元类别。该方案概述了使用改良狂犬病毒选择性标记神经元的方法。G 缺失狂犬病毒在立体定向注射到感兴趣的目标脑结构后，就像一种逆行示踪剂，并作为在神经元中递送和表达 EGFP 的载体。使用该技术可感染大量神经元，并使其整个树突都表达 GFP，从而产生单个神经元的“高尔基样”完整填充。因此，病毒介导的逆行追踪方法通过产生完整的细胞内填充，改进了传统的基于染料的逆行追踪技术。选择研究的脑区所有区域中分离良好的单个神经元进行重建，以获得具有代表性的神经元样本。该方案概述了重建跨越多个组织切片的标记神经元的细胞体和完整树突分支模式的程序。提取包括每个神经元在脑结构中的位置在内的形态学数据进行进一步分析。利用标准编程功能基于形态学指标进行独立聚类分析和聚类评估。为了验证这些分析的实用性，对在猕猴丘脑网状核（TRN）中重建的 160 个神经元进行的聚类分析进行了统计评估。此处进行的原始聚类分析和统计评估均表明，TRN 神经元可分为三个亚群，每个亚群都具有独特的形态特征。
