The cytoarchitectonic landscape revealed by deep learning method facilitated precise positioning in mouse neocortex.

Neocortex is a complex structure with different cortical sublayers and regions. However, the precise positioning of cortical regions can be challenging due to the absence of distinct landmarks without special preparation. To address this challenge, we developed a cytoarchitectonic landmark identification pipeline. The fluorescence micro-optical sectioning tomography method was employed to image the whole mouse brain stained by general fluorescent nucleotide dye. A fast 3D convolution network was subsequently utilized to segment neuronal somas in entire neocortex. By approach, the cortical cytoarchitectonic profile and the neuronal morphology were analyzed in 3D, eliminating the influence of section angle. And the distribution maps were generated that visualized the number of neurons across diverse morphological types, revealing the cytoarchitectonic landscape which characterizes the landmarks of cortical regions, especially the typical signal pattern of barrel cortex. Furthermore, the cortical regions of various ages were aligned using the generated cytoarchitectonic landmarks suggesting the structural changes of barrel cortex during the aging process. Moreover, we observed the spatiotemporally gradient distributions of spindly neurons, concentrated in the deep layer of primary visual area, with their proportion decreased over time. These findings could improve structural understanding of neocortex, paving the way for further exploration with this method.

Link Brain-Wide Projectome to Neuronal Dynamics in the Mouse Brain.

Knowledge about the neuronal dynamics and the projectome are both essential for understanding how the neuronal network functions in concert. However, it remains challenging to obtain the neural activity and the brain-wide projectome for the same neurons, especially for neurons in subcortical brain regions. Here, by combining in vivo microscopy and high-definition fluorescence micro-optical sectioning tomography, we have developed strategies for mapping the brain-wide projectome of functionally relevant neurons in the somatosensory cortex, the dorsal hippocampus, and the substantia nigra pars compacta. More importantly, we also developed a strategy to achieve acquiring the neural dynamic and brain-wide projectome of the molecularly defined neuronal subtype. The strategies developed in this study solved the essential problem of linking brain-wide projectome to neuronal dynamics for neurons in subcortical structures and provided valuable approaches for understanding how the brain is functionally organized via intricate connectivity patterns.

Single-neuron analysis of axon arbors reveals distinct presynaptic organizations between feedforward and feedback projections.

The morphology and spatial distribution of axon arbors and boutons are crucial for neuron presynaptic functions. However, the principles governing their whole-brain organization at the single-neuron level remain unclear. We developed a machine-learning method to separate axon arbors from passing axons in single-neuron reconstruction from fluorescence micro-optical sectioning tomography imaging data and obtained 62,374 axon arbors that displayed distinct morphology, spatial patterns, and scaling laws dependent on neuron types and targeted brain areas. Focusing on the axon arbors in the thalamus and cortex, we revealed the segregated spatial distributions and distinct morphology but shared topographic gradients between feedforward and feedback projections. Furthermore, we uncovered an association between arbor complexity and microglia density. Finally, we found that the boutons on terminal arbors show branch-specific clustering with a log-normal distribution that again differed between feedforward and feedback terminal arbors. Together, our study revealed distinct presynaptic structural organizations underlying diverse functional innervation of single projection neurons.

3D Visualization of Whole Brain Vessels and Quantification of Vascular Pathology in a Chronic Hypoperfusion Model Causing White Matter Damage.

Chronic cerebral hypoperfusion is an important pathological factor in many neurodegenerative diseases, such as cerebral small vessel disease (CSVD). One of the most used animal models for chronic cerebral hypoperfusion is the bilateral common carotid artery stenosis (BCAS) mouse. For the therapy of CSVD and other diseases, it will be beneficial to understand the pathological alterations of the BCAS mouse, particularly vascular pathological changes. A mouse model of BCAS was used, and 8 weeks later, cognitive function of the mice was examined by using novel object recognition test and eight-arm radial maze test. 11.7 T magnetic resonance imaging (MRI) and luxol fast blue staining were used to evaluate the injury of the corpus callosum (CC), anterior commissure (AC), internal capsule (IC), and optic tract (Opt) in the cerebral white matter of mice. Three-dimensional vascular images of the whole brain of mice were acquired using fluorescence micro-optical sectioning tomography (fMOST) with a high resolution of 0.32 × 0.32 × 1.00 µm3. Then, the damaged white matter regions were further extracted to analyze the vessel length density, volume fraction, tortuosity, and the number of vessels of different internal diameters. The mouse cerebral caudal rhinal vein was also extracted and analyzed for its branch number and divergent angle in this study. BCAS modeling for 8 weeks resulted in impaired spatial working memory, reduced brain white matter integrity, and myelin degradation in mice, and CC showed the most severe white matter damage. 3D revascularization of the whole mouse brain showed that the number of large vessels was reduced and the number of small vessels was increased in BCAS mice. Further analysis revealed that the vessel length density and volume fraction in the damaged white matter region of BCAS mice were significantly reduced, and the vascular lesions were most noticeable in the CC. At the same time, the number of small vessels in the above white matter regions was significantly reduced, while the number of microvessels was significantly increased in BCAS mice, and the vascular tortuosity was also significantly increased. In addition, the analysis of caudal rhinal vein extraction revealed that the number of branches and the average divergent angle in BCAS mice were significantly reduced. The BCAS modeling for 8 weeks will lead to vascular lesions in whole brain of mice, and the caudal nasal vein was also damaged, while BCAS mice mainly mitigated the damages by increasing microvessels. What is more, the vascular lesions in white matter of mouse brain can cause white matter damage and spatial working memory deficit. These results provide evidence for the vascular pathological alterations caused by chronic hypoperfusion.

Whole-brain mapping of histaminergic projections in mouse brain.

Histamine is a conserved neuromodulator in mammalian brains and critically involved in many physiological functions. Understanding the precise structure of the histaminergic network is the cornerstone in elucidating its function. Herein, using histidine decarboxylase (HDC)-CreERT2 mice and genetic labeling strategies, we reconstructed a whole-brain three dimensional (3D) structure of histaminergic neurons and their outputs at 0.32 × 0.32 × 2 µm3 pixel resolution with a cutting-edge fluorescence microoptical sectioning tomography system. We quantified the fluorescence density of all brain areas and found that histaminergic fiber density varied significantly among brain regions. The density of histaminergic fiber was positively correlated with the amount of histamine release induced by optogenetic stimulation or physiological aversive stimulation. Lastly, we reconstructed a fine morphological structure of 60 histaminergic neurons via sparse labeling and uncovered the largely heterogeneous projection pattern of individual histaminergic neurons. Collectively, this study reveals an unprecedented whole-brain quantitative analysis of histaminergic projections at the mesoscopic level, providing a foundation for future functional histaminergic study.

Multiscale reconstruction of bronchus and cancer cells in human lung adenocarcinoma.

BACKGROUND: While previous studies primarily focused on the structure of the normal whole mouse lung, the whole bronchus and cytoarchitectural details of the mouse intact lung lobe have been discovered at single-cell resolution. Revealing the sophisticated lung adenocarcinoma structure at three-dimensional (3D) and single-cell level remains a fundamental and critical challenge for the pathological mechanism research of lung adenocarcinoma (LA). METHODS: Fluorescence micro-optical Sectioning Tomography (fMOST) combined with PI staining were used to obtain the 3D imaging of the human LA tissue at single-cell resolution. RESULTS: With a spatial resolution of 0.32 × 0.32 × 1.0 µm3, the dataset of human LA with single-cell precision consists of two channels, each of which contains information about the bronchi and the cytoarchitecture. The bronchial wall is thicker and the lumen is smaller in the cancer tissue, in which its original normal structure is vanished. More solid components, more clustered cancer cells with larger nucleoli, and more significant atypia are found in cancer tissue. In paracancerous tissue, the bronchial wall cells have a monolayer or bilayer structure, cluster along the wall, and are relatively dispersed. Few fibrous structures and occasional dissemination of spread through air spaces (STAS) are observed. CONCLUSIONS: Based on the human LA tissue dataset obtained by fMOST and PI staining, the bronchi and cells were reconstructed and visualized. This work provides a technical roadmap for studying the bronchus and cytoarchitectural structure and their spatial relationship in LA tissue, which may help with the understanding of the main histological structure of LA among pathologists.

Whole-brain mapping of histaminergic projections in mouse brain / 中国药理学与毒理学杂志

OBJECTIVE Histamine is a conserved neuromodulator in mammalian brains and critically involved in many physiological functions.Understanding the precise structure of histaminergic network is the cor-nerstone in elucidating its function.METHODS Herein,using novel HDC-CreERT2 mice and genetic labeling strategies,we reconstructed a whole brain 3D structure of histaminergic neurons and their outputs at 0.32×0.32×2 μm3 pixel resolution with a cutting-edge fluorescence micro-optical sectioning tomography system(fMOST).And we dissect an appetite control circuit originating from the TMN to medial septal nucleus(MS)using fiber photometry,optogenetics,and chemogenetics interfer-ence.RESULTS We quantified the fluorescence density of all brain areas and found that histaminergic fiber density varied significantly among brain regions.The density of histaminergic fiber was positively correlated with the amount of histamine release induced by optogenetic stim-ulation or physiological aversive stimulation.Moreover,we reconstructed fine morphological structure of 60 hista-minergic neurons via sparse labeling,and uncovered the largely heterogeneous projection pattern of individual his-taminergic neuron.Lastly,we found that MS-projecting histaminergic circuit is functionally inhibited during food consumption,and bidirectionally modulates feeding behavior via downstream H2,but not H1,receptors on MS glutamatergic neurons.CONCLUSION Collectively,this study reveals an unprecedented whole-brain quanti-tative analysis of histaminergic projections at the meso-scopic level,providing a foundation for future functional histaminergic study.And we also demonstrate that this MS-projecting histaminergic circuit is critically involved in feeding,and H2Rs in MS glutamatergic neurons is a promising target for treating body weight problems.

Mapping the Function of Whole-Brain Projection at the Single Neuron Level.

Axonal projection conveys neural information. The divergent and diverse projections of individual neurons imply the complexity of information flow. It is necessary to investigate the relationship between the projection and functional information at the single neuron level for understanding the rules of neural circuit assembly, but a gap remains due to a lack of methods to map the function to whole-brain projection. Here an approach is developed to bridge two-photon calcium imaging in vivo with high-resolution whole-brain imaging based on sparse labeling with the genetically encoded calcium indicator GCaMP6. Reliable whole-brain projections are captured by the high-definition fluorescent micro-optical sectioning tomography (HD-fMOST). A cross-modality cell matching is performed and the functional annotation of whole-brain projection at the single-neuron level (FAWPS) is obtained. Applying it to the layer 2/3 (L2/3) neurons in mouse visual cortex, the relationship is investigated between functional preferences and axonal projection features. The functional preference of projection motifs and the correlation between axonal length in MOs and neuronal orientation selectivity, suggest that projection motif-defined neurons form a functionally specific information flow, and the projection strength in specific targets relates to the information clarity. This pipeline provides a new way to understand the principle of neuronal information transmission.

Multiscale Analysis of Cellular Composition and Morphology in Intact Cerebral Organoids.

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.

A spatial and cellular distribution of rabies virus infection in the mouse brain revealed by fMOST and single-cell RNA sequencing.

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.

Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing.

The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

Single-cell morphological characterization of CRH neurons throughout the whole mouse brain.

BACKGROUND: Corticotropin-releasing hormone (CRH) is an important neuromodulator that is widely distributed in the brain and plays a key role in mediating stress responses and autonomic functions. While the distribution pattern of fluorescently labeled CRH-expressing neurons has been studied in different transgenic mouse lines, a full appreciation of the broad diversity of this population and local neural connectivity can only come from integration of single-cell morphological information as a defining feature. However, the morphologies of single CRH neurons and the local circuits formed by these neurons have not been acquired at brain-wide and dendritic-scale levels. RESULTS: We screened the EYFP-expressing CRH-IRES-Cre;Ai32 mouse line to reveal the morphologies of individual CRH neurons throughout the whole mouse brain by using a fluorescence micro-optical sectioning tomography (fMOST) system. Diverse dendritic morphologies and projection fibers of CRH neurons were found in various brain regions. Follow-up reconstructions showed that hypothalamic CRH neurons had the smallest somatic volumes and simplest dendritic branches and that CRH neurons in several brain regions shared a common bipolar morphology. Further investigations of local CRH neurons in the medial prefrontal cortex unveiled somatic depth-dependent morphologies of CRH neurons that exhibited three types of mutual connections: basal dendrites (upper layer) with apical dendrites (layer 3); dendritic-somatic connections (in layer 2/3); and dendritic-dendritic connections (in layer 4). Moreover, hypothalamic CRH neurons were classified into two types according to their somatic locations and characteristics of dendritic varicosities. Rostral-projecting CRH neurons in the anterior parvicellular area had fewer and smaller dendritic varicosities, whereas CRH neurons in the periventricular area had more and larger varicosities that were present within dendrites projecting to the third ventricle. Arborization-dependent dendritic spines of CRH neurons were detected, among which the most sophisticated types were found in the amygdala and the simplest types were found in the hypothalamus. CONCLUSIONS: By using the CRH-IRES-Cre;Ai32 mouse line and fMOST imaging, we obtained region-specific morphological distributions of CRH neurons at the dendrite level in the whole mouse brain. Taken together, our findings provide comprehensive brain-wide morphological information of stress-related CRH neurons and may facilitate further studies of the CRH neuronal system.

Whole-Brain Reconstruction of Neurons in the Ventral Pallidum Reveals Diverse Projection Patterns.

The ventral pallidum (VP) integrates reward signals to regulate cognitive, emotional, and motor processes associated with motivational salience. Previous studies have revealed that the VP projects axons to many cortical and subcortical structures. However, descriptions of the neuronal morphologies and projection patterns of the VP neurons at the single neuron level are lacking, thus hindering the understanding of the wiring diagram of the VP. In this study, we used recently developed progress in robust sparse labeling and fluorescence micro-optical sectioning tomography imaging system (fMOST) to label mediodorsal thalamus-projecting neurons in the VP and obtain high-resolution whole-brain imaging data. Based on these data, we reconstructed VP neurons and classified them into three types according to their fiber projection patterns. We systematically compared the axonal density in various downstream centers and analyzed the soma distribution and dendritic morphologies of the various subtypes at the single neuron level. Our study thus provides a detailed characterization of the morphological features of VP neurons, laying a foundation for exploring the neural circuit organization underlying the important behavioral functions of VP.

Genetic Single Neuron Anatomy Reveals Fine Granularity of Cortical Axo-Axonic Cells.

Parsing diverse nerve cells into biological types is necessary for understanding neural circuit organization. Morphology is an intuitive criterion for neuronal classification and a proxy of connectivity, but morphological diversity and variability often preclude resolving the granularity of neuron types. Combining genetic labeling with high-resolution, large-volume light microscopy, we established a single neuron anatomy platform that resolves, registers, and quantifies complete neuron morphologies in the mouse brain. We discovered that cortical axo-axonic cells (AACs), a cardinal GABAergic interneuron type that controls pyramidal neuron (PyN) spiking at axon initial segments, consist of multiple subtypes distinguished by highly laminar-specific soma position and dendritic and axonal arborization patterns. Whereas the laminar arrangements of AAC dendrites reflect differential recruitment by input streams, the laminar distribution and local geometry of AAC axons enable differential innervation of PyN ensembles. This platform will facilitate genetically targeted, high-resolution, and scalable single neuron anatomy in the mouse brain.