To decipher the organizational logic of complex brain circuits, it is important to chart long-distance pathways while preserving micron-level accuracy of local network. However, mapping the neuronal projections with individual-axon resolution in the large and complex primate brain is still challenging. Herein, we describe a highly efficient pipeline for three-dimensional mapping of the entire macaque brain with subcellular resolution. The pipeline includes a novel poly-N-acryloyl glycinamide (PNAGA)-based embedding method for long-term structure and fluorescence preservation, high-resolution and rapid whole-brain optical imaging, and image post-processing. The cytoarchitectonic information of the entire macaque brain was acquired with a voxel size of 0.32 μm × 0.32 μm × 10 μm, showing its anatomical structure with cell distribution, density, and shape. Furthermore, thanks to viral labeling, individual long-distance projection axons from the frontal cortex were for the first time reconstructed across the entire brain hemisphere with a voxel size of 0.65 μm × 0.65 μm × 3 μm. Our results show that individual cortical axons originating from the prefrontal cortex simultaneously target multiple brain regions, including the visual cortex, striatum, thalamus, and midbrain. This pipeline provides an efficient method for cellular and circuitry investigation of the whole macaque brain with individual-axon resolution, and can shed light on brain function and disorders.

为了破译复杂大脑回路的组织逻辑，重要的是绘制长距离路径，同时保持本地网络的微米级精度。然而，在大型复杂的灵长类动物大脑中用个体轴突分辨率映射神经元投射仍然具有挑战性。在此，我们描述了一种高效的管道，用于以亚细胞分辨率对整个猕猴大脑进行三维映射。该管道包括一种基于聚-N-丙烯酰甘氨酰胺 (PNAGA) 的新型嵌入方法，用于长期结构和荧光保存、高分辨率和快速全脑光学成像以及图像后处理。获取了整个猕猴大脑的细胞结构信息，体素大小为0.32 μm × 0.32 μm × 10 μm，显示了其解剖结构，包括细胞分布、密度、和形状。此外，由于病毒标记，来自额叶皮层的单个长距离投射轴突首次在整个大脑半球重建，体素大小为 0.65 μm × 0.65 μm × 3 μm。我们的研究结果表明，源自前额叶皮层的单个皮层轴突同时靶向多个大脑区域，包括视觉皮层、纹状体、丘脑和中脑。该管道为具有个体轴突分辨率的整个猕猴大脑的细胞和电路研究提供了一种有效的方法，并且可以阐明大脑功能和疾病。我们的研究结果表明，源自前额叶皮层的单个皮层轴突同时靶向多个大脑区域，包括视觉皮层、纹状体、丘脑和中脑。该管道为具有个体轴突分辨率的整个猕猴大脑的细胞和电路研究提供了一种有效的方法，并且可以阐明大脑功能和疾病。我们的研究结果表明，源自前额叶皮层的单个皮层轴突同时靶向多个大脑区域，包括视觉皮层、纹状体、丘脑和中脑。该管道为具有个体轴突分辨率的整个猕猴大脑的细胞和电路研究提供了一种有效的方法，并且可以阐明大脑功能和疾病。