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A new method, named array fluorescent micro-optical sectioning tomography (array-fMOST), is developed for identifying the three-dimensional information at single-cell resolution from multi-samples.

Abstract Background Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. Methods In this study, we explore the use of fluorescence micro‐optical sectioning tomography (fMOST) and single‐cell RNA sequencing (scRNA‐seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of a recombinant RABV encoding enhanced green fluorescent protein (EGFP) under different infection routes, and a three‐dimensional (3D) view of RABV distribution in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA‐seq. Results Our fMOST data provided the 3D view of a neurotropic virus in the whole mouse brain, which indicated that the spatial distribution of RABV in the brain was influenced by the infection route. Interestingly, we provided evidence that RABV could infect multiple nuclei related to fear independent of different infection routes. More surprisingly, our scRNA‐seq data revealed that besides neurons RABV could infect macrophages and the infiltrating macrophages played at least three different antiviral roles during RABV infection. Conclusion This study draws a comprehensively spatial and cellular map of typical neurotropic virus infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of RABV and other neurotropic viruses.

The brain modulates specific functions in its various regions. Understanding the organization of different cells in the whole brain is crucial for investigating brain functions. Previous studies have focused on several regions and have had difficulty analyzing serial tissue samples. In this study, we introduced a pipeline to acquire anatomical and histological information quickly and efficiently from serial sections. First, we developed a serial brain-slice-staining method to stain serial sections and obtained more than 98.5% of slices with high integrity. Subsequently, using the self-developed analysis software, we registered and quantified the signals of imaged sections to the Allen Mouse Brain Common Coordinate Framework, which is compatible with multimodal images and slant section planes. Finally, we validated the pipeline with immunostaining by analyzing the activity variance in the whole brain during acute stress in aging and young mice. By removing the problems resulting from repeated manual operations, this pipeline is widely applicable to serial brain slices from multiple samples in a rapid and convenient manner, which benefits to facilitate research in life sciences.

Cerebral organoids recapitulate in vivo phenotypes and physiological functions of the brain and have great potential in studying brain development, modeling diseases, and conducting neural network research. It is essential to obtain whole-mount three-dimensional (3D) images of cerebral organoids at cellular levels to explore their characteristics and applications. Existing histological strategies sacrifice inherent spatial characteristics of organoids, and the strategy for volume imaging and 3D analysis of entire organoids is urgently needed. Here, we proposed a high-resolution imaging pipeline based on fluorescent labeling by viral transduction and 3D immunostaining with fluorescence micro-optical sectioning tomography (fMOST). We were able to image intact organoids using our pipeline, revealing cytoarchitecture information of organoids and the spatial localization of neurons and glial fibrillary acidic protein positive cells (GFAP+ cells). We performed single-cell reconstruction to analyze the morphology of neurons and GFAP+ cells. Localization and quantitative analysis of cortical layer markers revealed heterogeneity of organoids. This pipeline enabled acquisition of high-resolution spatial information of millimeter-scale organoids for analyzing their cell composition and morphology.

Axonopathy is a pathological feature observed in both Alzheimer’s disease (AD) patients and animal models. However, identifying the temporal and regional progression of axonopathy during AD development remains elusive. Using the fluorescence micro-optical sectioning tomography system, we acquired whole-brain datasets in the early stage of 5xFAD/Thy1-GFP-M mice. We reported that among GFP labeled axons, GFP-positive axonopathy first formed in the lateral septal nucleus, subiculum, and medial mammillary nucleus. The axonopathy further increased in most brain regions during aging. However, most of the axonopathic varicosities disappeared significantly in the medial mammillary nucleus after 8 weeks old. Continuous three-dimensional datasets showed that axonopathy in the medial mammillary nucleus was mainly located on axons from hippocampal GFP-positive neurons. Using the rabies viral tracer in combination with immunohistochemistry, we found that axons in the medial mammillary nucleus from the subiculum were susceptible to lesions that prior to the occurrence of behavioral disorders. In conclusion, we created an early-stage spatiotemporal map of axonopathy in 5xFAD/Thy1-GFP-M mice and identified specific neural circuits which are vulnerable to axon lesions in an AD mouse model. These findings underline the importance of early interventions for AD, and may contribute to the understanding of its progression and its early symptom treatment.

Mapping the cytoarchitecture of the whole brain can reveal the organizational logic of neural systems. However, this remains a significant challenge, especially for gyrencephalic brains with a large volume. Here we propose an integrated pipeline for generating a cytoarchitectonic atlas with single-cell resolution of the whole brain. To analyze a large-volume brain, we used a modified en-bloc Nissl staining protocol to achieve uniform staining of large-scale brain specimens from ferret (Mustela putorius furo). By combining whole-brain imaging and big data processing, we established strategies for parsing cytoarchitectural information at a voxel resolution of 0.33 μm × 0.33 μm × 1 μm and terabyte-scale data analysis. Using the cytoarchitectonic datasets for adult ferret brain, we identified giant pyramidal neurons in ferret brains and provide the first report of their morphological diversity, neurochemical phenotype, and distribution patterns in the whole brain in three dimensions. This pipeline will facilitate studies on the organization and development of the mammalian brains, from that of rodents to the gyrencephalic brains of ferret and even primates.

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.

Study Design. Retrospective comparative study. Objective. To investigate the occurrence of neurological complications in patients undergoing thoracic three-column osteotomy (3CO) utilizing an magnetic resonance imaging (MRI)-based classification that assesses spinal cord shape and the presence of cerebrospinal fluid at the curve apex and evaluate its prognostic capacity for postoperative neurological deficits. Summary of Background Data. Recent advancements in correction techniques have improved outcomes for severe spinal deformity patients undergoing 3CO. A novel MRI-based spinal cord classification system was introduced, but its validation and association with postoperative complications remain unexplored. Materials and Methods. Between September 2012 and September 2018, a retrospective analysis was conducted on 158 adult patients with spinal deformities undergoing 3CO. Radiographic parameters were measured. T2-weighted axial MRI was used to describe spinal cord morphology at the apex. Intraoperative neurophysiological monitoring alerts were recorded, and preoperative and postoperative neurological functions were assessed using the Frankel score. Categorical data were compared using the χ² or the Fisher exact test. The paired t test was utilized to assess the mean difference between preoperative and postoperative measurements, while the one-way analysis of variance and independent t test were used for comparative analyses among the different spinal cord types. Results. Patients were categorized into three groups: type 1, type 2, and type 3, consisting of 12, 85, and 61 patients. Patients with type 3 morphology exhibited larger Cobb angles of the main curve (P<0.001). This disparity persisted both postoperatively and during follow-up (P< 0.05). Intraoperative neurophysiological monitoring alerts were triggered in 32 patients (20.3%), with a distribution of one case in type 1, six cases in type 2, and 22 cases in type 3 morphologies (P<0.001). New neurological deficits were observed in 15 patients (9.5%), with 1, 3, and 11 cases in type 1, 2, and 3 morphologies, respectively. Conclusions. Patients with type 3 morphology exhibited greater spinal deformity severity, a higher likelihood of preoperative neurological deficits, and an elevated risk of postoperative neurological complications. This underscores the utility of the classi- fication as a tool for predicting postoperative neurological complications in patients undergoing thoracic 3CO. Level of Evidence: 4. [ABSTRACT FROM AUTHOR]