Virtual reality-empowered deep-learning analysis of brain cells.

Automated detection of specific cells in three-dimensional datasets such as whole-brain light-sheet image stacks is challenging. Here, we present DELiVR, a virtual reality-trained deep-learning pipeline for detecting c-Fos+ cells as markers for neuronal activity in cleared mouse brains. Virtual reality annotation substantially accelerated training data generation, enabling DELiVR to outperform state-of-the-art cell-segmenting approaches. Our pipeline is available in a user-friendly Docker container that runs with a standalone Fiji plugin. DELiVR features a comprehensive toolkit for data visualization and can be customized to other cell types of interest, as we did here for microglia somata, using Fiji for dataset-specific training. We applied DELiVR to investigate cancer-related brain activity, unveiling an activation pattern that distinguishes weight-stable cancer from cancers associated with weight loss. Overall, DELiVR is a robust deep-learning tool that does not require advanced coding skills to analyze whole-brain imaging data in health and disease.

Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

Whole-body cellular mapping in mouse using standard IgG antibodies.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-ß-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php .

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Mapping of neuroinflammation-induced hypoxia in the spinal cord using optoacoustic imaging.

Recent studies suggest that metabolic changes and oxygen deficiency in the central nervous system play an important role in the pathophysiology of multiple sclerosis (MS). In our present study, we investigated the changes in oxygenation and analyzed the vascular perfusion of the spinal cord in a rodent model of MS. We performed multispectral optoacoustic tomography of the lumbar spinal cord before and after an oxygen enhancement challenge in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. In addition, mice were transcardially perfused with lectin to label the vasculature and their spinal columns were optically cleared, followed by light sheet fluorescence microscopy. To analyze the angioarchitecture of the intact spine, we used VesSAP, a novel deep learning-based framework. In EAE mice, the spinal cord had lower oxygen saturation and hemoglobin concentration compared to healthy mice, indicating compromised perfusion of the spinal cord. Oxygen administration reversed hypoxia in the spinal cord of EAE mice, although the ventral region remained hypoxic. Additionally, despite the increased vascular density, we report a reduction in length and complexity of the perfused vascular network in EAE. Taken together, these findings highlight a new aspect of neuroinflammatory pathology, revealing a significant degree of hypoxia in EAE in vivo that is accompanied by changes in spinal vascular perfusion. The study also introduces optoacoustic imaging as a tractable technique with the potential to further decipher the role of hypoxia in EAE and to monitor it in MS patients.

A guidebook for DISCO tissue clearing.

Histological analysis of biological tissues by mechanical sectioning is significantly time-consuming and error-prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue-clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands-on implementation.

Cell Surface Protein mRNAs Show Differential Transcription in Pyramidal and Fast-Spiking Cells as Revealed by Single-Cell Sequencing.

The prefrontal cortex (PFC) plays a key role in higher order cognitive functions and psychiatric disorders such as autism, schizophrenia, and depression. In the PFC, the two major classes of neurons are the glutamatergic pyramidal (Pyr) cells and the GABAergic interneurons such as fast-spiking (FS) cells. Despite extensive electrophysiological, morphological, and pharmacological studies of the PFC, the therapeutically utilized drug targets are restricted to dopaminergic, glutamatergic, and GABAergic receptors. To expand the pharmacological possibilities as well as to better understand the cellular and network effects of clinically used drugs, it is important to identify cell-type-selective, druggable cell surface proteins and to link developed drug candidates to Pyr or FS cell targets. To identify the mRNAs of such cell-specific/enriched proteins, we performed ultra-deep single-cell mRNA sequencing (19 685 transcripts in total) on electrophysiologically characterized intact PFC neurons harvested from acute brain slices of mice. Several selectively expressed transcripts were identified with some of the genes that have already been associated with cellular mechanisms of psychiatric diseases, which we can now assign to Pyr (e.g., Kcnn2, Gria3) or FS (e.g., Kcnk2, Kcnmb1) cells. The earlier classification of PFC neurons was also confirmed at mRNA level, and additional markers have been provided.

Deep learning-enabled multi-organ segmentation in whole-body mouse scans.

Whole-body imaging of mice is a key source of information for research. Organ segmentation is a prerequisite for quantitative analysis but is a tedious and error-prone task if done manually. Here, we present a deep learning solution called AIMOS that automatically segments major organs (brain, lungs, heart, liver, kidneys, spleen, bladder, stomach, intestine) and the skeleton in less than a second, orders of magnitude faster than prior algorithms. AIMOS matches or exceeds the segmentation quality of state-of-the-art approaches and of human experts. We exemplify direct applicability for biomedical research for localizing cancer metastases. Furthermore, we show that expert annotations are subject to human error and bias. As a consequence, we show that at least two independently created annotations are needed to assess model performance. Importantly, AIMOS addresses the issue of human bias by identifying the regions where humans are most likely to disagree, and thereby localizes and quantifies this uncertainty for improved downstream analysis. In summary, AIMOS is a powerful open-source tool to increase scalability, reduce bias, and foster reproducibility in many areas of biomedical research.

Machine learning analysis of whole mouse brain vasculature.

Tissue clearing methods enable the imaging of biological specimens without sectioning. However, reliable and scalable analysis of large imaging datasets in three dimensions remains a challenge. Here we developed a deep learning-based framework to quantify and analyze brain vasculature, named Vessel Segmentation & Analysis Pipeline (VesSAP). Our pipeline uses a convolutional neural network (CNN) with a transfer learning approach for segmentation and achieves human-level accuracy. By using VesSAP, we analyzed the vascular features of whole C57BL/6J, CD1 and BALB/c mouse brains at the micrometer scale after registering them to the Allen mouse brain atlas. We report evidence of secondary intracranial collateral vascularization in CD1 mice and find reduced vascularization of the brainstem in comparison to the cerebrum. Thus, VesSAP enables unbiased and scalable quantifications of the angioarchitecture of cleared mouse brains and yields biological insights into the vascular function of the brain.

Cellular and Molecular Probing of Intact Human Organs.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex.

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer's disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Retino-cortical stimulus frequency-dependent gamma coupling: evidence and functional implications of oscillatory potentials.

Long-range gamma band EEG oscillations mediate information transmission between distant brain regions. Gamma band-based coupling may not be restricted to cortex-to-cortex communication but may include extracortical parts of the visual system. The retinogram and visual event-related evoked potentials exhibit time-locked, forward propagating oscillations that are candidates of gamma oscillatory coupling between the retina and the visual cortex. In this study, we tested if this gamma coupling is present as indicated by the coherence of gamma-range (70-200 Hz) oscillatory potentials (OPs) recorded simultaneously from the retina and the primary visual cortex in freely moving, adult rats. We found significant retino-cortical OP coherence in a wide range of stimulus duration (0.01-1000 msec), stimulus intensity (800-5000 mcd/mm2), interstimulus interval (10-400 msec), and stimulus frequency (0.25-25 Hz). However, at low stimulus frequencies, the OPs were time-locked, flickering light at 25 Hz entrained continuous OP coherence (steady-state response, SSR). Our results suggest that the retina and the visual cortex exhibit oscillatory coupling at high-gamma frequency with precise time locking and synchronization of information transfer from the retina to the visual cortex, similar to cortico-cortical gamma coupling. The temporal fusion of retino-cortical gamma coherence at stimulus rates of theater movies may explain the mechanism of the visual illusion of continuity. How visual perception depends on early transformations of ascending sensory information is incompletely understood. By simultaneous measurement of flash-evoked potentials in the retina and the visual cortex in awake, freely moving rats, we demonstrate for the first time that time-locked gamma oscillatory potentials exhibit stable retino-cortical synchrony across a wide range of stimulus parameters and that the temporal continuity of coherence changes with stimulus frequency according to the expected change in the visual illusion of continuity.

Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.