Abstract：
In this novel large-scale multiplexed immunofluorescence study we comprehensively characterized and compared layer-specific proteomic features within regions of interest of the widely divergent dorsolateral prefrontal cortex (A46) and primary visual cortex (A17) of adult rhesus monkeys. Twenty-eight markers were imaged in rounds of sequential staining, and their spatial distribution precisely quantified within gray matter layers and superficial white matter. Cells were classified as neurons, astrocytes, oligodendrocytes, microglia, or endothelial cells. The distribution of fibers and blood vessels were assessed by quantification of staining intensity across regions of interest. This method revealed multivariate similarities and differences between layers and areas. Protein expression in neurons was the strongest determinant of both laminar and regional differences, whereas protein expression in glia was more important for intra-areal laminar distinctions. Among specific results, we observed a lower glia-to-neuron ratio in A17 than in A46 and the pan-neuronal markers HuD and NeuN were differentially distributed in both brain areas with a lower intensity of NeuN in layers 4 and 5 of A17 compared to A46 and other A17 layers. Astrocytes and oligodendrocytes exhibited distinct marker-specific laminar distributions that differed between regions; notably, there was a high proportion of ALDH1L1-expressing astrocytes and of oligodendrocyte markers in layer 4 of A17. The many nuanced differences in protein expression between layers and regions observed here highlight the need for direct assessment of proteins, in addition to RNA expression, and set the stage for future protein-focused studies of these and other brain regions in normal and pathological conditions.
摘要：
在这项新的大规模多重免疫荧光研究中，我们全面表征和比较了成年恒河猴的广泛发散的背外侧前额叶皮层（A46）和初级视觉皮层（A17）的感兴趣区域内的层特异性蛋白质组特征。在连续染色的轮次中对28个标记进行了成像，它们的空间分布在灰质层和浅层白质中精确量化。细胞被分类为神经元，星形胶质细胞,少突胶质细胞,小胶质细胞,或内皮细胞。通过对感兴趣区域的染色强度进行定量来评估纤维和血管的分布。该方法揭示了层和区域之间的多变量相似性和差异。神经元中的蛋白质表达是层状和区域差异的最强决定因素，而神经胶质中的蛋白质表达对于区域内层状差异更为重要。在具体结果中，我们观察到A17的神经胶质细胞与神经元的比率低于A46，而泛神经元标记HuD和NeuN在两个大脑区域中的差异分布，与A46和其他A17层相比，A17的第4层和第5层的NeuN强度较低。星形胶质细胞和少突胶质细胞表现出不同的标记特异性层状分布，这些层状分布在区域之间不同；值得注意的是，在A17的第4层中，表达ALDH1L1的星形胶质细胞和少突胶质细胞标志物的比例很高。这里观察到的层和区域之间蛋白质表达的许多细微差别突出了直接评估蛋白质的必要性。除了RNA表达，并为未来在正常和病理条件下对这些和其他大脑区域进行以蛋白质为重点的研究奠定了基础。
