The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei.

Understanding amazingly complex brain functions and pathologies requires a complete cerebral vascular atlas in stereotaxic coordinates. Making a precise atlas for cerebral arteries and veins has been a century-old objective in neuroscience and neuropathology. Using micro-optical sectioning tomography (MOST) with a modified Nissl staining method, we acquired five mouse brain data sets containing arteries, veins, and microvessels. Based on the brain-wide vascular spatial structures and brain regions indicated by cytoarchitecture in one and the same mouse brain, we reconstructed and annotated the vascular system atlas of both arteries and veins of the whole mouse brain for the first time. The distributing patterns of the vascular system within the brain regions were acquired and our results show that the patterns of individual vessels are different from each other. Reconstruction and statistical analysis of the microvascular network, including derivation of quantitative vascular densities, indicate significant differences mainly in vessels with diameters less than 8 μm and large than 20 μm across different brain regions. Our precise cerebral vascular atlas provides an important resource and approach for quantitative studies of brain functions and diseases.

Corticotropin-releasing hormone (CRH), with widespread expression in the brain, plays a key role in modulating a series of behaviors, including anxiety, arousal, motor function, learning and memory. Previous studies have focused on some brain regions with densely distributed CRH neurons such as paraventricular hypothalamic nucleus (PVH) and bed nuclei of the stria terminalis (BST) and revealed some basic structural and functional knowledge of CRH neurons. However, there is no systematic analysis of brain-wide distribution of CRH neurons. Here, we performed a comprehensive study of CRH neurons in CRH-IRES-Cre;Ai3 mice via automatic imaging and stereoscopic cell counting in a whole mouse brain. We acquired four datasets of the CRH distributions with co-localized cytoarchitecture at a voxel resolution of 0.32 μm × 0.32 μm × 2 μm using brain-wide positioning system (BPS). Next, we precisely located and counted the EYFP-labeled neurons in different regions according to propidium iodide counterstained anatomical reference using Neuronal Global Position System. In particular, dense EYFP expression was found in piriform area, BST, central amygdalar nucleus, PVH, Barrington’s nucleus, and inferior olivary complex. Considerable CRH neurons were also found in main olfactory bulb, medial preoptic nucleus, pontine gray, tegmental reticular nucleus, external cuneate nucleus, and midline thalamus. We reconstructed and compared the soma morphology of CRH neurons in 11 brain regions. The results demonstrated that CRH neurons had regional diversities of both cell distribution and soma morphology. This anatomical knowledge enhances the current understanding of the functions of CRH neurons. These results also demonstrated the ability of our platform to accurately orient, reconstruct and count neuronal somas in three-dimension for type-specific neurons in the whole brain, making it feasible to answer the fundamental neuroscience question of exact numbers of various neurons in the whole brain.