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Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

Abstract Background The medial prefrontal cortex (mPFC) is involved in complex functions containing multiple types of neurons in distinct subregions with preferential roles. The pyramidal neurons had wide-range projections to cortical and subcortical regions with subregional preferences. Using a combination of viral tracing and fluorescence micro-optical sectioning tomography (fMOST) in transgenic mice, we systematically dissected the whole-brain connectomes of intratelencephalic (IT) and pyramidal tract (PT) neurons in four mPFC subregions. Results IT and PT neurons of the same subregion projected to different target areas while receiving inputs from similar upstream regions with quantitative differences. IT and PT neurons all project to the amygdala and basal forebrain, but their axons target different subregions. Compared to subregions in the prelimbic area (PL) which have more connections with sensorimotor-related regions, the infralimbic area (ILA) has stronger connections with limbic regions. The connection pattern of the mPFC subregions along the anterior–posterior axis showed a corresponding topological pattern with the isocortex and amygdala but an opposite orientation correspondence with the thalamus. Conclusions By using transgenic mice and fMOST imaging, we obtained the subregional preference whole-brain connectomes of IT and pyramidal tract PT neurons in the mPFC four subregions. These results provide a comprehensive resource for directing research into the complex functions of the mPFC by offering anatomical dissections of the different subregions.

This systematic review aims to explore published research on the use of ChatGPT in language learning between November 2022 and November 2023, outlining the types of papers, methodologies adopted, publishing journals, major research trends, topics of interest, and existing gaps demanding attention. The PRISMA framework was utilized to capture the latest published articles, selecting 36 articles that met the inclusion criteria. Findings extracted from this review include (1) authors worldwide contribute to this topic, with Asia and North America leading; (2) the wide distribution across various journals underscores the interdisciplinary nature of this research topic, such as computer science, psychology, linguistics, education, and other social sciences; (3) empirical research dominates the literature that is published, with the majority focusing on higher education and ethical considerations. Other findings include that ChatGPT plays multifaceted roles, supporting self-directed language learning, content generation, and teacher workflows. Research gaps include the need for diversified scopes, longitudinal studies, exploration of stakeholders’ perceptions, and assessments of feedback quality.

This study appraises the ideological criticism that emerged in the Chinese architectural sphere during the anti-waste campaign of 1955, which marks a pivotal moment in the history of modern Chinese architecture. Within this timeframe, architects and architectural scholars such as Chen Gan, Shui Fengman, and Liu Dunzhen, respectively questioned, defended, and criticized Liang Sicheng’s architectural ideas. Their articles were published in prominent Chinese publications including People’s Daily, Wenyi Bao [Art News], Jianzhu Xuebao [Journal of Architecture], and Xin Jianshe [Journal of New Construction]. By delving deep into the conceptual framework and the logical structure of these long-neglected critique articles, this study unveils the intricate debates in Chinese architectural practice and theoretical concepts during the 1950s. The findings highlight that the ideological critique during the anti-waste campaign not only reflects the infiltration of politicized rhetoric into the architectural domain but also assumes a profound significance in terms of the early attempts of Chinese architects to re-evaluate architectural historiography through the lens of Marxist theory. This pivotal event laid the foundation for the gradual formation and consolidation of a series of discursive patterns within the Chinese architectural community, exerting a lasting influence on contemporary architectural discussions and writings in multifarious ways.

In multicolor fluorescence microscopy, it is crucial to orient biological structures at a single-cell resolution based on precise anatomical annotations of cytoarchitecture images. However, during synchronous multicolor imaging, due to spectral mixing, the crosstalk from the blue signals of 4′,6-diamidino-2-phenylindole (DAPI)-stained cytoarchitecture images to the green waveband hinders the visualization and identification of green signals. Here, we proposed a deep learning-based framework named the crosstalk elimination and cytoarchitecture enhancement pipeline (CECEP) to simultaneously acquire crosstalk-free signals in the green channel and high-contrast DAPI-stained cytoarchitecture images during multicolor fluorescence imaging. For the CECEP network, we proposed an unsupervised learning algorithm named the cytoarchitecture enhancement network (CENet), which increased the signal-to-background ratio (SBR) of the cytoarchitecture images from 1.5 to 15.0 at a reconstruction speed of 25 Hz for 1800 × 1800 pixel images. The CECEP network is widely applicable to images of different quality, different types of tissues, and different multicolor fluorescence microscopy. In addition, the CECEP network can also facilitate various downstream analysis tasks, such as cell recognition, structure tensor calculation, and brain region segmentation. With the CECEP network, we simultaneously acquired two specific fluorescence-labeled neuronal distributions and their colocated high-SBR cytoarchitecture images without crosstalk throughout the brain. Experimental results demonstrate that our method could potentially facilitate multicolor fluorescence imaging applications in biology, such as revealing and visualizing different types of biological structures with precise locations and orientations.

Cytoarchitecture, the organization of cells within organs and tissues, serves as a crucial anatomical foundation for the delineation of various regions. It enables the segmentation of the cortex into distinct areas with unique structural and functional characteristics. While traditional 2D atlases have focused on cytoarchitectonic mapping of cortical regions through individual sections, the intricate cortical gyri and sulci demands a 3D perspective for unambiguous interpretation. In this study, we employed fluorescent micro-optical sectioning tomography to acquire architectural datasets of the entire macaque brain at a resolution of 0.65 μm × 0.65 μm × 3 μm. With these volumetric data, the cortical laminar textures were remarkably presented in appropriate view planes. Additionally, we established a stereo coordinate system to represent the cytoarchitectonic information as surface-based tomograms. Utilizing these cytoarchitectonic features, we were able to three-dimensionally parcel the macaque cortex into multiple regions exhibiting contrasting architectural patterns. The whole-brain analysis was also conducted on mice that clearly revealed the presence of barrel cortex and reflected biological reasonability of this method. Leveraging these high-resolution continuous datasets, our method offers a robust tool for exploring the organizational logic and pathological mechanisms of the brain’s 3D anatomical structure.

Ieoh Ming Pei completed his work on the Fragrant Hill Hotel in Beijing in the year of 1983. This has been a piece of significant work in modern Chinese architecture. Corresponding studies and discussions have been mainly attracted to its style and its exploration of modernity, while relatively less concentrated on a close reading of the design. Taking “site reconstruction” as a clue, this paper traces the historic transformations of the site condition of this hotel. Following a chronological review and a formal analysis of different time periods, the paper revisits the discourse of the Fragrant Hill Hotel both in Chinese and American contexts. It examines the gaps between Chinese and American critiques and speculates on the reasons for the discrepancies. By comparing the discursive focus of “hunting park” and “imperial garden,” this paper attempts to uncover the tension between history and identity concealed in I.M. Pei’s address on the site. While the inconsistency is partly due to the time and people’s standpoints originating from the time, it still shows how design functions as a form of power in the history.

The brain–computer interface (BCI) is one of the most powerful tools in neuroscience and generally includes a recording system, a processor system, and a stimulation system. Optogenetics has the advantages of bidirectional regulation, high spatiotemporal resolution, and cell-specific regulation, which expands the application scenarios of BCIs. In recent years, optogenetic BCIs have become widely used in the lab with the development of materials and software. The systems were designed to be more integrated, lightweight, biocompatible, and power efficient, as were the wireless transmission and chip-level embedded BCIs. The software is also constantly improving, with better real-time performance and accuracy and lower power consumption. On the other hand, as a cutting-edge technology spanning multidisciplinary fields including molecular biology, neuroscience, material engineering, and information processing, optogenetic BCIs have great application potential in neural decoding, enhancing brain function, and treating neural diseases. Here, we review the development and application of optogenetic BCIs. In the future, combined with other functional imaging techniques such as near-infrared spectroscopy (fNIRS) and functional magnetic resonance imaging (fMRI), optogenetic BCIs can modulate the function of specific circuits, facilitate neurological rehabilitation, assist perception, establish a brain-to-brain interface, and be applied in wider application scenarios.

Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer’s disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.

Abstract Background Type 2 Diabetes mellitus (T2DM) has become a major lifestyle disease endangering human health worldwide. Patients with T2DM face varying degrees of loneliness, which adversely affects their family and the larger society. This study investigates the serial multiple mediating roles of depression and self-perceived burden between family function and loneliness in the T2DM population of China. Methods In total, 260 T2DM patients were included. They rated themselves based on UCLA Loneliness Scale, Self-Rating Depression Scale, Self-Rating Anxiety Scale, Family Care Index, and Self-Perceived Burden Scale. Pearson and Spearman correlation analyses were conducted to clarify the association among variables. The SPSS macro-PROCESS program was used for a series of multiple mediation analyses. Results Family function, depression, self-perceived burden, and loneliness were significantly correlated (P

Abstract Background Based on their anatomical location, rostral projections of nuclei are classified as ascending circuits, while caudal projections are classified as descending circuits. Upper brainstem neurons participate in complex information processing and specific sub-populations preferentially project to participating ascending or descending circuits. Cholinergic neurons in the upper brainstem have extensive collateralizations in both ascending and descending circuits; however, their single-cell projection patterns remain unclear because of the lack of comprehensive characterization of individual neurons. Results By combining fluorescent micro-optical sectional tomography with sparse labeling, we acquired a high-resolution whole-brain dataset of pontine-tegmental cholinergic neurons (PTCNs) and reconstructed their detailed morphology using semi-automatic reconstruction methods. As the main source of acetylcholine in some subcortical areas, individual PTCNs had abundant axons with lengths up to 60 cm and 5000 terminals and innervated multiple brain regions from the spinal cord to the cortex in both hemispheres. Based on various collaterals in the ascending and descending circuits, individual PTCNs were grouped into four subtypes. The morphology of cholinergic neurons in the pedunculopontine nucleus was more divergent, whereas the laterodorsal tegmental nucleus neurons contained richer axonal branches and dendrites. In the ascending circuits, individual PTCNs innervated the thalamus in three different patterns and projected to the cortex via two separate pathways. Moreover, PTCNs targeting the ventral tegmental area and substantia nigra had abundant collaterals in the pontine reticular nuclei, and these two circuits contributed oppositely to locomotion. Conclusions Our results suggest that individual PTCNs have abundant axons, and most project to various collaterals in the ascending and descending circuits simultaneously. They target regions with multiple patterns, such as the thalamus and cortex. These results provide a detailed organizational characterization of cholinergic neurons to understand the connexional logic of the upper brainstem.

The sensorimotor cortex participates in diverse functions with different reciprocally connected subregions and projection-defined pyramidal neuron types therein, while the fundamental organizational logic of its circuit elements at the single-cell level is still largely unclear. Here, using mouse Cre driver lines and high-resolution whole-brain imaging to selectively trace the axons and dendrites of cortical pyramidal tract (PT) and intratelencephalic (IT) neurons, we reconstructed the complete morphology of 1,023 pyramidal neurons and generated a projectome of 6 subregions within the sensorimotor cortex. Our morphological data revealed substantial hierarchical and layer differences in the axonal innervation patterns of pyramidal neurons. We found that neurons located in the medial motor cortex had more diverse projection patterns than those in the lateral motor and sensory cortices. The morphological characteristics of IT neurons in layer 5 were more complex than those in layer 2/3. Furthermore, the soma location and morphological characteristics of individual neurons exhibited topographic correspondence. Different subregions and layers were composed of different proportions of projection subtypes that innervate downstream areas differentially. While the axonal terminals of PT neuronal population in each cortical subregion were distributed in specific subdomains of the superior colliculus (SC) and zona incerta (ZI), single neurons selectively innervated a combination of these projection targets. Overall, our data provide a comprehensive list of projection types of pyramidal neurons in the sensorimotor cortex and begin to unveil the organizational principle of these projection types in different subregions and layers.

The main olfactory bulb is the key element of the olfactory pathway of rodents. To precisely dissect the neural pathway in the main olfactory bulb (MOB), it is necessary to construct the three-dimensional morphologies of the anatomical structures within it with micro-level resolution. However, the construction remains challenging due to the complicated shape of the anatomical structures in the main olfactory bulb and the high resolution of micro-optical images. To address these issues, we propose an interactive volume image segmentation method with micro-level resolution in the horizontal and axial direction. Firstly, we obtain the initial location of the anatomical structures by manual annotation and design a patch-based neural network to learn the complex texture feature of the anatomical structures. Then we randomly sample some patches to predict by the trained network and perform an annotation reconstruction based on intensity calculation to get the final location results of the anatomical structures. Our experiments were conducted using Nissl-stained brain images acquired by the Micro-optical sectioning tomography (MOST) system. Our method achieved a mean dice similarity coefficient (DSC) of 81.8% and obtain the best segmentation performance. At the same time, the experiment shows the three-dimensional morphology reconstruction results of the anatomical structures in the main olfactory bulb are smooth and consistent with their natural shapes, which addresses the possibility of constructing three-dimensional morphologies of the anatomical structures in the whole brain.

Understanding the developmental changes that affect neurons is a key step in exploring the assembly and maturation of neural circuits in the brain. For decades, researchers have used a number of labeling techniques to visualize neuronal morphology at different stages of development. However, the efficiency and accuracy of neuronal labeling technologies are limited by the complexity and fragility of neonatal brains. In this review, we illustrate the various labeling techniques utilized for examining the neurogenesis and morphological changes occurring during the early stages of development. We compare the advantages and limitations of each technique from different aspects. Then, we highlight the gaps remaining in our understanding of the structure of neurons in the neonatal mouse brain.

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: Nuclear factor erythroid-derived 2-like 2 (Nrf2) is the master regulator of antioxidant defenses. High-intensity interval training (HIIT) has been proposed as a time-efficient training program and has become a substantial component of modern training program. In the present study, we evaluated the effects of sulforaphane (SFN), a dietary isothiocyanate derived from cruciferous vegetables and a potent Nrf2 activator, on Nrf2-mediated antioxidant defense responses of skeletal muscle induced by exhaustive exercise in HIIT mice. Male C57BL/6J mice were randomly allocated into control group, HIIT group, and HIIT pretreated with SFN (HIIT+SFN) group. On the third day after completion of a 6-weeks HIIT protocol, an exhaustive treadmill test was conducted in all mice. Mice were intraperitoneally injected with SFN (HIIT+SFN group) or PBS (HIIT and control mice) 4 times in 3 days prior to the exhaustive treadmill test. The results indicated that the 6-weeks HIIT protocol did not increase the antioxidative capacity of skeletal muscle during exhaustive exercise. Importantly, SFN treatment improved antioxidative capacity of skeletal muscle in response to the acute exhaustive exercise by increasing mRNA and nucleoprotein expression of Nrf2 and these genes involved in antioxidant generation and decreasing blood creatine kinase (CK) and 4-hydroxy-2-nonenal (4-HNE)-modified protein levels in the HIIT mice.

Vascular segmentation is a crucial task in biomedical image processing, which is significant for analyzing and modeling vascular networks under physiological and pathological states. With advances in fluorescent labeling and mesoscopic optical techniques, it has become possible to map the whole-mouse-brain vascular networks at capillary resolution. However, segmenting vessels from mesoscopic optical images is a challenging task. The problems, such as vascular signal discontinuities, vessel lumens, and background fluorescence signals in mesoscopic optical images, belong to global semantic information during vascular segmentation. Traditional vascular segmentation methods based on convolutional neural networks (CNNs) have been limited by their insufficient receptive fields, making it challenging to capture global semantic information of vessels and resulting in inaccurate segmentation results. Here, we propose SegVesseler, a vascular segmentation method based on Swin Transformer. SegVesseler adopts 3D Swin Transformer blocks to extract global contextual information in 3D images. This approach is able to maintain the connectivity and topology of blood vessels during segmentation. We evaluated the performance of our method on mouse cerebrovascular datasets generated from three different labeling and imaging modalities. The experimental results demonstrate that the segmentation effect of our method is significantly better than traditional CNNs and achieves state-of-the-art performance.

Summary: The dorsal bed nucleus of stria terminalis (dBNST) is a pivotal hub for stress response modulation. Dysfunction of dopamine (DA) network is associated with chronic stress, but the roles of DA network of dBNST in chronic stress-induced emotional disorders remain unclear. We examine the role of dBNST Drd1+ and Drd2+ neurons in post-weaning social isolation (PWSI)-induced behavior deficits. We find that male, but not female, PWSI rats exhibit negative emotional phenotypes and the increase of excitability and E-I balance of dBNST Drd2+ neurons. More importantly, hypofunction of dBNST Drd2 receptor underlies PWSI-stress-induced male-specific neuronal plasticity change of dBNST Drd2+ neurons. Furthermore, chemogenetic activation of dBNST Drd2+ neurons is sufficient to induce anxiogenic effects, while Kir4.1-mediated chronic inhibition of dBNST Drd2+ neurons ameliorate PWSI-induced anxiety-like behaviors. Our findings reveal an important neural mechanism underlying PWSI-induced sex-specific behavioral abnormalities and potentially provide a target for the treatment of social stress-related emotional disorder.

Summary: Resin embedding combined with ultra-thin sectioning has been widely used in microscopic and electron imaging to acquire precise structural information of biological tissues. However, the existing embedding method was detrimental to quenchable fluorescent signals of precise structures and pH-insensitive fluorescent dyes. Here, we developed a low-temperature chemical polymerization method named HM20-T to maintain weak signals of various precise structures and to decrease background fluorescence. The fluorescence preservation ratio of green fluorescent protein (GFP) tagged presynaptic elements and tdTomato labeled axons doubled. The HM20-T method was suitable for a variety of fluorescent dyes, such as DyLight 488 conjugated Lycopersicon esculentum lectin. Moreover, the brains also retained immunoreactivity after embedding. In summary, the HM20-T method was suitable for the characterization of multi-color labeled precise structures, which would contribute to the acquisition of complete morphology of various biological tissues and to the investigation of composition and circuit connection in the whole brain.

Summary: The neocortex mediates information processing through highly organized circuitry that contains various neuron types. Distinct populations of projection neurons in different cortical regions and layers make specific connections and participate in distinct physiological functions. Herein, with the fluorescence micro-optical sectioning tomography (fMOST) and transgenetic mice that targeted intratelencephalic (IT) and pyramidal tract (PT) neurons at specific layers, we dissected the long-range connectome of pyramidal neurons in six subregions of the sensorimtor cortex. The distribution of the input neurons indicated that IT and PT neurons in the same region received information from similar regions, while the neurons in different subregions received from the preferred neuron populations. Both the input and projection areas of these six subregions showed the transverse and longitudinal correspondence in the cortico-cortical, cortico-thalamic, and cortico-striatal circuits, which indicated that the connections were topologically organized. This study provides a comprehensive resource on the anatomical connections of cortical circuits.

High-definition fluorescent micro-optical sectioning tomography is employed to resolve fine structures of multiple vessel types in the intact liver.

Organoids can recapitulate human-specific phenotypes and functions in vivo and have great potential for research in development, disease modeling, and drug screening. Due to the inherent variability among organoids, experiments often require a large sample size. Embedding, staining, and imaging each organoid individually require a lot of reagents and time. Hence, there is an urgent need for fast and efficient methods for analyzing the phenotypic changes in organoids in batches. Here, we provide a comprehensive strategy for array embedding, staining, and imaging of cerebral organoids in both agarose sections and in 3D to analyze the spatial distribution of biomarkers in organoids in situ. We constructed several disease models, particularly an aging model, as examples to demonstrate our strategy for the investigation of the phenotypic analysis of organoids. We fabricated an array mold to produce agarose support with microwells, which hold organoids in place for live/dead imaging. We performed staining and imaging of sectioned organoids embedded in agarose and 3D imaging to examine phenotypic changes in organoids using fluorescence micro-optical sectioning tomography (fMOST) and whole-mount immunostaining. Parallel studies of organoids in arrays using the same staining and imaging parameters enabled easy and reliable comparison among different groups. We were able to track all the data points obtained from every organoid in an embedded array. This strategy could help us study the phenotypic changes in organoids in disease models and drug screening.

Short-term memory deficits are associated with prefrontal cortex dysfunction in Alzheimer’s disease. Here, the authors assessed extratelencephalic projection (ET) neurons and found reduced ET neural activity in the medial prefrontal cortex (mPFC) and showed ET neurons received fewer cholinergic inputs from the basal forebrain in 5×FAD mice which led to object recognition memory deficits.

Abstract 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.

Visualizing the relationships and interactions among different biological components in the whole brain is crucial to our understanding of brain structures and functions. However, an automatic multicolor whole-brain imaging technique is still lacking. Here, we developed a multicolor wide-field large-volume tomography (multicolor WVT) to simultaneously acquire fluorescent signals in blue, green, and red channels in the whole brain. To facilitate the segmentation of brain regions and anatomical annotation, we used 4′, 6-diamidino-2-phenylindole (DAPI) to provide cytoarchitecture through real-time counterstaining. We optimized the imaging planes and modes of three channels to overcome the axial chromatic aberration of the illumination path and avoid the crosstalk from DAPI to the green channel without the modification of system configuration. We also developed an automatic contour recognition algorithm based on DAPI-staining cytoarchitecture to shorten data acquisition time and reduce data redundancy. To demonstrate the potential of our system in deciphering the relationship of the multiple components of neural circuits, we acquired and quantified the brain-wide distributions of cholinergic neurons and input of ventral Caudoputamen (CP) with the anatomical annotation in the same brain. We further identified the cholinergic type of upstream neurons projecting to CP through the triple-color collocated analysis and quantified its proportions in the two brain-wide distributions. Both accounted for 0.22%, implying CP might be modulated by non-cholinergic neurons. Our method provides a new research tool for studying the different biological components in the same organ and potentially facilitates the understanding of the processing mechanism of neural circuits and other biological activities.

Compared to young people and adults, there are differences in the ability of elderly people to resist diseases or injuries, with some noticeable features being gender-dependent. However, gender differences in age-related viscera alterations are not clear. To evaluate a potential possibility of gender differences during the natural aging process, we used three age groups to investigate the impact on spleens, kidneys, and adrenal glands. The immunofluorescence results showed that male-specific p21 proteins were concentrated in the renal tubule epithelial cells of the kidney. Histological staining revealed an increase in the frequencies of fat vacuoles located in the renal tubule epithelial cells of the cortex, under the renal capsule in the kidneys of male mice with age. In female mice, we found that the width of the globular zone in the adrenal gland cortex was unchanged with age. On the contrary, the male displayed a reduction in width. Compared to females, the content of epinephrine in adrenal gland tissue according to ELISA analysis was higher in adults, and a greater decline was observed in aged males particularly. These data confirmed the age-dependent differences between female and male mice; therefore, gender should be considered one of the major factors for personalized treatment in clinical diagnosis and treatment.

Waterfront intervention, as one of the post-industrial reuse paradigms, has flourished around the world and been studied as a global phenomenon. This paper investigates the application of a dual adaptive reuse strategy to industrial heritage waterfront buildings and explores its social significance. The case study is of the West Bund, a waterfront renovation in Shanghai, China. Insights are drawn from the qualitative research approach of triangulation, with evidence derived from document sources, archival records, direct participants and semi-structured interviews. This paper examines a series of galleries and landscapes created from former industrial buildings and facilities along the Huangpu River. It concludes that the West Bund exemplifies a dual strategy of adaptive reuse: art-led and landscape-led building reuse. This dual strategy can be viewed as an endeavour to balance urban gentrification and publicness in the megalopolis. It can also be argued, however, that the dual approach to adaptive reuse is unsustainable within the framework of an entrepreneurial state, and significantly so in the post-epidemic era when economic growth pressure increases. This study contributes to our understanding of the complex nature of industrial heritage in the rapidly shifting landscape of contemporary China.

The pontomesencephalic tegmentum, comprising the pedunculopontine nucleus and laterodorsal tegmental nucleus, is involved in various functions via complex connections; however, the organizational structure of these circuits in the whole brain is not entirely clear. Here, combining viral tracing with fluorescent micro-optical sectional tomography, we comprehensively investigated the input and output circuits of two cholinergic subregions in a continuous whole-brain dataset. We found that these nuclei receive abundant input with similar spatial distributions but with different quantitative measures and acquire similar neuromodulatory afferents from the ascending reticular activation system. Meanwhile, these cholinergic nuclei project to similar targeting areas throughout multiple brain regions and have different spatial preferences in 3D. Moreover, some cholinergic connections are unidirectional, including projections from the pedunculopontine nucleus and laterodorsal tegmental nucleus to the ventral posterior complex of the thalamus, and have different impacts on locomotion and anxiety. These results reveal the integrated cholinergic connectome of the midbrain, thus improving the present understanding of the organizational structure of the pontine-tegmental cholinergic system from its anatomical structure to its functional modulation.

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.

Centrifugal projections in the olfactory system are critical to both olfactory processing and behavior. The olfactory bulb (OB), the first relay station in odor processing, receives a substantial number of centrifugal inputs from the central brain regions. However, the anatomical organization of these centrifugal connections has not been fully elucidated, especially for the excitatory projection neurons of the OB, the mitral/tufted cells (M/TCs). Using rabies virus-mediated retrograde monosynaptic tracing in Thy1-Cre mice, we identified that the three most prominent inputs of the M/TCs came from the anterior olfactory nucleus (AON), the piriform cortex (PC), and the basal forebrain (BF), similar to the granule cells (GCs), the most abundant population of inhibitory interneurons in the OB. However, M/TCs received proportionally less input from the primary olfactory cortical areas, including the AON and PC, but more input from the BF and contralateral brain regions than GCs. Unlike organizationally distinct inputs from the primary olfactory cortical areas to these two types of OB neurons, inputs from the BF were organized similarly. Furthermore, individual BF cholinergic neurons innervated multiple layers of the OB, forming synapses on both M/TCs and GCs. Taken together, our results indicate that the centrifugal projections to different types of OB neurons may provide complementary and coordinated strategies in olfactory processing and behavior.

Malignant melanoma, an increasingly common form of skin cancer, is a major threat to public health, especially when the disease progresses past skin lesions to the stage of advanced metastasis. Targeted drug development is an effective strategy for the treatment of malignant melanoma. In this work, a new antimelanoma tumor peptide, the lebestatin–annexin V (designated LbtA5) fusion protein, was developed and synthesized by recombinant DNA techniques. As a control, annexin V (designated ANV) was also synthesized by the same method. The fusion protein combines annexin V, which specifically recognizes and binds phosphatidylserine, with the disintegrin lebestatin (lbt), a polypeptide that specifically recognizes and binds integrin α1β1. LbtA5 was successfully prepared with good stability and high purity while retaining the dual biological activity of ANV and lbt. MTT assays demonstrated that both ANV and LbtA5 could reduce the viability of melanoma B16F10 cells, but the activity of the fusion protein LbtA5 was superior to that of ANV. The tumor volume growth was slowed in a mouse xenograft model treated with ANV and LbtA5, and the inhibitory effect of high concentrations of LbtA5 was significantly better than that of the same dose of ANV and was comparable to that of DTIC, a drug used clinically for melanoma treatment. The hematoxylin and eosin (H&E) staining test showed that ANV and LbtA5 had antitumor effects, but LbtA5 showed a stronger ability to induce melanoma necrosis in mice. Immunohistochemical experiments further showed that ANV and LbtA5 may inhibit tumor growth by inhibiting angiogenesis in tumor tissue. Fluorescence labeling experiments showed that the fusion of ANV with lbt enhanced the targeting of LbtA5 to mouse melanoma tumor tissue, and the amount of target protein in tumor tissue was significantly increased. In conclusion, effective coupling of the integrin α1β1-specific recognition molecule lbt confers stronger biological antimelanoma effects of ANV, which may be achieved by the dual effects of effective inhibition of B16F10 melanoma cell viability and inhibition of tumor tissue angiogenesis. The present study describes a new potential strategy for the application of the promising recombinant fusion protein LbtA5 in the treatment of various cancers, including malignant melanoma.

The feedback control system with network-connected components is vulnerable to cyberattacks. We study a problem of attack detection in supervisory control of discrete-event systems. The scenario of a system subjected to actuator enablement attacks is considered in this article. We also consider that some unsafe places that should be protected from an attacker exist in the system, and some controllable events that are disabled by a supervisor might be re-enabled by an attacker. This article proposes a defense strategy to detect actuator enablement attacks and disable all controllable events after detecting an attack. We design algorithmic procedures to determine whether the system can be protected against damage caused by actuator enablement attacks, where the damage is predefined as a set of “unsafe” places. In this way, the system property is called “AE-safe controllability”. The safe controllability can be verified by using a basis diagnoser or a basis verifier. Finally, we explain the approach with a cargo system example.

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.

The glutamatergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) mediated diverse brain functions. However, their whole-brain neural connectivity has not been comprehensively mapped. Here we used the virus tracers to characterize the whole-brain inputs and outputs of glutamatergic and GABAergic neurons in VTA and SNc. We found that these neurons received similar inputs from upstream brain regions, but some quantitative differences were also observed. Neocortex and dorsal striatum provided a greater share of input to VTA glutamatergic neurons. Periaqueductal gray and lateral hypothalamic area preferentially innervated VTA GABAergic neurons. Specifically, superior colliculus provided the largest input to SNc glutamatergic neurons. Compared to input patterns, the output patterns of glutamatergic and GABAergic neurons in the VTA and SNc showed significant preference to different brain regions. Our results laid the anatomical foundation for understanding the functions of cell-type-specific neurons in VTA and SNc.

The dorsal raphe nucleus (DR) and median raphe nucleus (MR) contain populations of glutamatergic and GABAergic neurons that regulate diverse behavioral functions. However, their whole-brain input-output circuits remain incompletely elucidated. We used viral tracing combined with fluorescence micro-optical sectioning tomography to generate a comprehensive whole-brain atlas of inputs and outputs of glutamatergic and GABAergic neurons in the DR and MR. We found that these neurons received inputs from similar upstream brain regions. The glutamatergic and GABAergic neurons in the same raphe nucleus had divergent projection patterns with differences in critical brain regions. Specifically, MR glutamatergic neurons projected to the lateral habenula through multiple pathways. Correlation and cluster analysis revealed that glutamatergic and GABAergic neurons in the same raphe nucleus received heterogeneous inputs and sent different collateral projections. This connectivity atlas further elucidates the anatomical architecture of the raphe nuclei, which could facilitate better understanding of their behavioral functions.

Tree resistance to overturning is crucial in forestry hazard applications and management. Tree anchorage varies considerably with species, tree age, and site conditions. We investigate the relationship between the root characteristics of the overturning slip surface and the role of roots (regarding different diameters in overturning). Four Leucaena leucocephala were fully excavated by a quadrate monolith to establish root distribution characteristics, and 19 L. leucocephala were uprooted until the trees completely overturned to measure the anchoring resistance to overturning. A model was developed to improve the descriptions of root characteristics in the mechanical processes for tree overturning. The results show that the distribution characteristics of the root system were well described by the model. For the root–soil plate radius, the thickest root diameter and the root biomass of different diameters at the overturning slip surface increased with the diameter at the breast height. The root biomass affected the strength of the overturning slip surface; the root density may be a key factor in identifying the location of the overturning slip surface. The model could predict the overturning moment of most overturned trees; although it overestimated the overturning moment for small diameters at breast height trees, the results will be useful for understanding the influence of root distribution characteristics in overturning.

Abstract Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

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.

This article explores how reconstruction design can be used in urban heritage as an adaptive approach to creating a continuous cultural dynamic for urban regeneration. The case that is studied is the rebuilding project of the Porcelain Tower of Nanjing, well-known to westerners as a splendour of the East. The research strategy stems from the epistemological paradigm of interpretivism and relies mainly on qualitative research methods. The ethic of refashioning a past legacy is discussed by reviewing the relevant theories and documents. It is argued that the rationale for rebuilding heritage should be judged on a case-by-case basis. From the perspective of urban cultural regeneration, rebuilding can be an appropriate solution to reviving heritage within the bounds of authenticity. Respecting the delicate balance between historical significance and contemporary sustainability, urban heritage can sometimes best be served by modern reconstruction. This paper, therefore, identifies the modern Porcelain Tower as an urban landmark that distinguishes Nanjing among Chinese cities and satisfies the cultural demand for sustainable local urban regeneration. Whilst the modern pagoda is not historically inaccurate, its reconstruction was determined without public engagement in a way that emphasises the government’s view of its history and value.

Recently, a lot of engineering constructions have been carried out in the Loess Plateau of China. However, the collapsibility of compacted loess as a filler poses a potential threat to the safety and stability of buildings. To address this threat, the scientific evaluation of the collapsibility of compacted loess has become a key to engineering construction. This study, therefore, tested the resistivity of compacted loess under varying conditions of water content, dry density, and its collapsibility coefficient under a pressure of 200 kPa. The correlation between collapsibility and electrical parameters was determined, and based on the resistivity, a new method to evaluate the collapsibility of the compacted loess is proposed. The results show that the resistivity of compacted loess decreases with an increase in water content and dry density and that the effect of water content is more significant. There is a critical water content value that causes the resistivity decay rate to slow down. The collapsibility coefficient also decreases with an increase in water content and dry density, and under the same dry density or same water content, the collapsibility coefficient and resistivity are positively correlated. According to the normalized resistivity value ρ/ρw, the relative collapsibility coefficient δs /0.015, and the water content ω obtained in the experiment, a collapsibility coefficient prediction model based on the resistivity of compacted loess was established. Using resistivity to evaluate the collapsibility of loess is nondestructive and provides a new method to accurately and quickly evaluate the collapsibility of compacted loess.

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.

Summary: Resin embedding is widely used to dissect the fine structure of bio-tissue with electron and optical microscopy. However, it is difficult to embed large-volume tissues with resin. Here, we modified the formula of LR-White resin to prevent the sample cracking during polymerization process and applied this method to the intact brains of mouse, ferret, and macaque. Meanwhile, we increased the fluorescence preservation rate for green fluorescent protein (GFP) from 73 ± 4.0% to 126 ± 3.0% and tdTomato from 60 ± 3.3% to 117 ± 2.8%. Combined with the whole-brain imaging system, we acquired the cytoarchitectonic information and the circuit information such as individual axon and boutons which were labeled with multiple fluorescent proteins. This method shows great potential in the study of continuous fine microstructure information in large-volume tissues from different species, which can facilitate the neuroscience research and help the understanding of the structure-function relationship in complex bio-tissues.

Abstract Tissue optical clearing techniques have provided important tools for large‐volume imaging. Aqueous‐based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m‐xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA‐based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.

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.

Summary: Ventral hippocampus (vHIP) and medial prefrontal cortex (mPFC) are both critical regions for social behaviors. However, how their interactions affect social behavior is not well understood. By viral tracing, optogenetics, chemogenetics, and fiber photometry, we demonstrated that inhibition of vHIP or direct projections from vHIP to mPFC impaired social memory expression. Via rabies retrograde tracing, we found that all three major GABAergic neurons in mPFC received direct inputs from vHIP. Activation of parvalbumin positive (PV+) neurons in mPFC but not somatostatin positive (SST+) neurons can rescue the social memory impairment caused by vHIP inhibition. Furthermore, fiber photometry results demonstrated that social behaviors preferentially recruited PV+ neurons and inhibition of hippocampal neurons disrupted the activity of PV+ neurons during social interactions. These results revealed a new mechanism of how vHIP and mPFC regulate social behavior in complementarity with the existing neural circuitry mechanism. : Behavioral Neuroscience; Neuroscience; Systems Neuroscience Subject Areas: Behavioral Neuroscience, Neuroscience, Systems Neuroscience

The segmentation of brain region contours in three dimensions is critical for the analysis of different brain structures, and advanced approaches are emerging continuously within the field of neurosciences. With the development of high-resolution micro-optical imaging, whole-brain images can be acquired at the cellular level. However, brain regions in microscopic images are aggregated by discrete neurons with blurry boundaries, the complex and variable features of brain regions make it challenging to accurately segment brain regions. Manual segmentation is a reliable method, but is unrealistic to apply on a large scale. Here, we propose an automated brain region segmentation framework, DeepBrainSeg, which is inspired by the principle of manual segmentation. DeepBrainSeg incorporates three feature levels to learn local and contextual features in different receptive fields through a dual-pathway convolutional neural network (CNN), and to provide global features of localization by image registration and domain-condition constraints. Validated on biological datasets, DeepBrainSeg can not only effectively segment brain-wide regions with high accuracy (Dice ratio > 0.9), but can also be applied to various types of datasets and to datasets with noises. It has the potential to automatically locate information in the brain space on the large scale.

To better understand and analyze the unsaturated stability of loess filling body, it is necessary to study the changes in suction stress before and after the drying-wetting cycles. In this study, the SWCC of compacted loess before and after drying-wetting cycles was tested using the filter paper method. Then, the suction stress was calculated and the microstructure of the loess sample was determined by the SEM and NMR. The results showed that the drying-wetting cycles had an important influence on the SSCC and microstructure of compacted loess. The change in suction stress before and after the drying-wetting cycles can be well explained by the loess microstructure. The drying-wetting cycles did not significantly change the basic trend of the compacted loess’s SSCC, but it increased the porosity and the dominant pore diameter of loess, and reduced the suction stress under the same matric suction. The main significant change in suction stress with matric suction occurred within the range of the dominant soil pores. The larger the dominant pore diameter, the smaller the suction stress under the same matric suction. In addition, this study proposes a new method for calculating suction stress based on the PSD parameters.

The excitatory neurons in the visual cortex are of great significance for us in understanding brain functions. However, the diverse neuron types and their morphological properties have not been fully deciphered. In this paper, we applied the brain-wide positioning system (BPS) to image the entire brain of two Thy1-eYFP H-line male mice at 0.2 μm × 0.2 μm × 1 μm voxel resolution. A total of 103 neurons were reconstructed in layers 5 and 6 of the visual cortex with single-axon-level resolution. Based on the complete topology of neurons and the inherent positioning function of the imaging method, we classified the observed neurons into six types according to their apical dendrites and somata location: star pyramidal cells in layer 5 (L5-sp), slender-tufted pyramidal cells in layer 5 (L5-st), tufted pyramidal cells in layer 5 (L5-tt), spiny stellate-like cells in layer 6 (L6-ss), star pyramidal cells in layer 6 (L6-sp), and slender-tufted pyramidal cells in layer 6 (L6-st). By examining the axonal projection patterns of individual neurons, they can be categorized into three modes: ipsilateral circuit connection neurons, callosal projection neurons and corticofugal projection neurons. Correlating the two types of classifications, we have found that there are at least two projection modes comprised in the former defined neuron types except for L5-tt. On the other hand, each projection mode may consist of four dendritic types defined in this study. The axon projection mode only partially correlates with the apical dendrite feature. This work has demonstrated a paradigm for resolving the visual cortex through single-neuron-level quantification and has shown potential to be extended to reveal the connectome of other defined sensory and motor systems.

Paraffin embedding is widely used in microscopic imaging for preparing biological specimens. However, owing to significant fluorescence quenching during the embedding process, it is not compatible with fluorescent-labeling techniques, such as transgenic and viral labeling using green fluorescent protein (GFP). Here, we investigate the quenching mechanism and optimize the embedding process to improve the preservation of fluorescence intensity. The results show that dehydration is the main reason for fluorescence quenching during paraffin embedding, caused by the full denaturation of GFP molecules in ethyl alcohol. To evaluate fluorescent and morphological preservation, we modified the embedding process using tertiary butanol (TBA) instead of ethyl alcohol. Fluorescence intensity following TBA dehydration increased 12.08-fold of that observed in the traditional method. We obtained uniform fluorescence maintenance throughout the whole mouse brain, while the continuous apical dendrites, spines, and axon terminals were shown evenly within the cortex, hippocampus, and the amygdala. Moreover, we embedded a whole rat brain labeled with AAV in the prelimbic cortex (Prl). With the axon terminals in different areas, such as the caudate putamen, thalamus, and pyramidal tract, the results showed a continuous tract of Prl neurons throughout the whole brain. This method was also suitable for tdTomota labeled samples. These findings indicate that this modified embedding method could be compatible with GFP and provides a potential turning point for applications in the fluorescent labeling of samples.

Long-range neuronal circuits play an important role in motor and sensory information processing. Determining direct synaptic inputs of excited and inhibited neurons is important for understanding the circuit mechanisms involved in regulating movement. Here, we used the monosynaptic rabies tracing technique, combined with fluorescent micro-optical sectional tomography, to characterize the brain-wide input to the motor cortex (MC). The whole brain dataset showed that the main excited and inhibited neurons in the MC received inputs from similar brain regions with a quantitative difference. With 3D reconstruction we found that the distribution of input neurons, that target the primary and secondary MC, had different patterns. In the cortex, the neurons projecting to the primary MC mainly distributed in the lateral and anterior portion, while those to the secondary MC distributed in the medial and posterior portion. The input neurons in the subcortical areas also showed the topographic shift model, as in the thalamus, the neurons distributed as outer and inner shells while the neurons in the claustrum and amygdala were in the ventral and dorsal part, respectively. These results lay the anatomical foundation to understanding the organized pattern of motor circuits and the functional differences between the primary and secondary MC.