ABSTRACT
Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.
Subject(s)
Molecular Imaging/methods , Tissue Preservation/methods , Algorithms , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Nerve Fibers, Myelinated/chemistry , Proteomics , Reducing Agents , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.
Subject(s)
Hydrogels/chemistry , Staining and Labeling/methods , Tissue Engineering/methods , Acrylamide/chemistry , Animals , Biomechanical Phenomena , Biomimetic Materials/chemistry , Bioprinting , Cerebral Cortex/chemistry , Cross-Linking Reagents/chemistry , Elastic Modulus , Hippocampus/chemistry , Humans , Materials Testing , Mice , Stress, Mechanical , Tensile StrengthABSTRACT
Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage of 3 kDa dextran (but not 2 MDa dextran) indicating microinfarction-related blood-brain barrier (BBB) dysfunction for small molecules. At day 5, the local edema disappeared with the partial restoration of microcirculation and recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes expressing glial fibrillary acidic protein (GFAP) appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct until 30 days, inducing glial scar formation in cerebral cortex. Our longitudinal intravital imaging of serial microscopic neurovascular pathophysiology in cerebral microinfarction newly revealed that astrocytes are critically susceptible to the acute microinfarction and their reactive response leads to the fibrous glial scar formation.
Subject(s)
Astrocytes , Gliosis , Animals , Astrocytes/metabolism , Dextrans/metabolism , Glial Fibrillary Acidic Protein/metabolism , Gliosis/diagnostic imaging , Gliosis/etiology , Gliosis/metabolism , Infarction/metabolism , Intravital Microscopy , MiceABSTRACT
Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso-ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.
Subject(s)
Brain/embryology , Cell Differentiation , Cell Proliferation , Organoids/growth & development , Body Patterning , Humans , Spatio-Temporal AnalysisABSTRACT
Systemic delivery of bioactive molecules in the CNS is hampered by the blood-brain barrier, which has bottlenecked noninvasive physiological study of the brain and the development of CNS drugs. Here we report that irradiation with an ultrashort pulsed laser to the blood vessel wall induces transient leakage of blood plasma without compromising vascular integrity. By combining this method with a systemic injection, we delivered target molecules in various tissues, including the brain cortex. This tool allows minimally invasive local delivery of chemical probes, nanoparticles, and viral vectors into the brain cortex. Furthermore, we demonstrated astrocyte-mediated vasodilation in vivo without opening the skull, using this method to load a calcium indicator in conjunction with label-free photoactivation of astrocytes.
Subject(s)
Brain/blood supply , Brain/metabolism , Capillary Permeability , Microscopy, Fluorescence/methods , Animals , Astrocytes/metabolism , Calcium/metabolism , Fluorescent Dyes/pharmacology , Lasers , Light , Male , Mice , Mice, Inbred ICR , Permeability , Photochemistry/methods , PhotonsABSTRACT
Circular RNAs (circRNAs) are noncoding RNAs abundant in brain tissue, and many are derived from activity-dependent, linear mRNAs encoding for synaptic proteins, suggesting that circRNAs may directly or indirectly play a role in regulating synaptic development, plasticity, and function. However, it is unclear if the circular forms of these RNAs are similarly regulated by activity and what role these circRNAs play in developmental plasticity. Here, we employed transcriptome-wide analysis comparing differential expression of both mRNAs and circRNAs in juvenile mouse primary visual cortex (V1) following monocular deprivation (MD), a model of developmental plasticity. Among the differentially expressed mRNAs and circRNAs following 3-day MD, the circular and the activity-dependent linear forms of the Homer1 gene, circHomer1 and Homer1a respectively, were of interest as their expression changed in opposite directions: circHomer1 expression increased while the expression of Homer1a decreased following MD. Knockdown of circHomer1 prevented the depression of closed-eye responses normally observed after 3-day MD. circHomer1-knockdown led to a reduction in average dendritic spine size prior to MD, but critically there was no further reduction after 3-day MD, consistent with impaired structural plasticity. circHomer1-knockdown also prevented the reduction of surface AMPA receptors after 3-day MD. Synapse-localized puncta of the AMPA receptor endocytic protein Arc increased in volume after MD but were smaller in circHomer1-knockdown neurons, suggesting that circHomer1 regulates plasticity through mechanisms of activity-dependent AMPA receptor endocytosis. Thus, activity-dependent circRNAs regulate developmental synaptic plasticity, and our findings highlight the essential role of circHomer1 in V1 plasticity induced by short-term MD.
ABSTRACT
The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.
Subject(s)
Gene Rearrangement , Translocation, Genetic , Humans , Animals , Mice , Mutation , Genomics , Chromosome Inversion , MammalsABSTRACT
The thalamus is the main gateway for sensory information from the periphery to the mammalian cerebral cortex. A major conundrum has been the discrepancy between the thalamus's central role as the primary feedforward projection system into the neocortex and the sparseness of thalamocortical synapses. Here we use new methods, combining genetic tools and scalable tissue expansion microscopy for whole-cell synaptic mapping, revealing the number, density and size of thalamic versus cortical excitatory synapses onto individual layer 2/3 (L2/3) pyramidal cells (PCs) of the mouse primary visual cortex. We find that thalamic inputs are not only sparse, but remarkably heterogeneous in number and density across individual dendrites and neurons. Most surprising, despite their sparseness, thalamic synapses onto L2/3 PCs are smaller than their cortical counterparts. Incorporating these findings into fine-scale, anatomically faithful biophysical models of L2/3 PCs reveals how individual neurons with sparse and weak thalamocortical synapses, embedded in small heterogeneous neuronal ensembles, may reliably 'read out' visually driven thalamic input.
Subject(s)
Neocortex , Thalamus , Mice , Animals , Thalamus/physiology , Neurons/physiology , Synapses/physiology , Pyramidal Cells , MammalsABSTRACT
Spatial transcriptomics is a newly emerging field that enables high-throughput investigation of the spatial localization of transcripts and related analyses in various applications for biological systems. By transitioning from conventional biological studies to "in situ" biology, spatial transcriptomics can provide transcriptome-scale spatial information. Currently, the ability to simultaneously characterize gene expression profiles of cells and relevant cellular environment is a paradigm shift for biological studies. In this review, recent progress in spatial transcriptomics and its applications in neuroscience and cancer studies are highlighted. Technical aspects of existing technologies and future directions of new developments (as of March 2023), computational analysis of spatial transcriptome data, application notes in neuroscience and cancer studies, and discussions regarding future directions of spatial multi-omics and their expanding roles in biomedical applications are emphasized.
Subject(s)
Neoplasms , Transcriptome , Transcriptome/genetics , Gene Expression Profiling , Neoplasms/genetics , Neoplasms/therapyABSTRACT
Autism spectrum disorders (ASD) represent neurodevelopmental disorders characterized by social deficits, repetitive behaviors, and various comorbidities, including epilepsy. ANK2, which encodes a neuronal scaffolding protein, is frequently mutated in ASD, but its in vivo functions and disease-related mechanisms are largely unknown. Here, we report that mice with Ank2 knockout restricted to cortical and hippocampal excitatory neurons (Ank2-cKO mice) show ASD-related behavioral abnormalities and juvenile seizure-related death. Ank2-cKO cortical neurons show abnormally increased excitability and firing rate. These changes accompanied decreases in the total level and function of the Kv7.2/KCNQ2 and Kv7.3/KCNQ3 potassium channels and the density of these channels in the enlengthened axon initial segment. Importantly, the Kv7 agonist, retigabine, rescued neuronal excitability, juvenile seizure-related death, and hyperactivity in Ank2-cKO mice. These results suggest that Ank2 regulates neuronal excitability by regulating the length of and Kv7 density in the AIS and that Kv7 channelopathy is involved in Ank2-related brain dysfunctions.
Subject(s)
Epilepsy , KCNQ Potassium Channels , Animals , Mice , Epilepsy/metabolism , KCNQ Potassium Channels/genetics , KCNQ2 Potassium Channel/genetics , KCNQ2 Potassium Channel/metabolism , KCNQ3 Potassium Channel/metabolism , Neurons/metabolism , Seizures/genetics , Seizures/metabolismABSTRACT
Synaptic NMDA receptors can produce powerful dendritic supralinearities that expand the computational repertoire of single neurons and their respective circuits. This form of supralinearity may represent a general principle for synaptic integration in thin dendrites. However, individual cortical neurons receive many diverse classes of input that may require distinct postsynaptic decoding schemes. Here, we show that sensory, motor, and thalamic inputs preferentially target basal, apical oblique, and distal tuft dendrites, respectively, in layer 5b pyramidal neurons of the mouse retrosplenial cortex, a visuospatial association area. These dendritic compartments exhibited differential expression of NMDA receptor-mediated supralinearity due to systematic changes in the AMPA-to-NMDA receptor ratio. Our results reveal a new schema for integration in cortical pyramidal neurons, in which dendrite-specific changes in synaptic receptors support input-localized decoding. This coexistence of multiple modes of dendritic integration in single neurons has important implications for synaptic plasticity and cortical computation.
Subject(s)
Pyramidal Cells , Receptors, N-Methyl-D-Aspartate , Animals , Cerebral Cortex/physiology , Dendrites/physiology , Mice , Pyramidal Cells/physiology , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/physiology , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic AcidABSTRACT
Synthetic tissue-hydrogel methods have enabled superresolution investigation of biological systems using diffraction-limited microscopy. However, chemical modification by fixatives can cause loss of antigenicity, limiting molecular interrogation of the tissue gel. Here, we present epitope-preserving magnified analysis of proteome (eMAP) that uses purely physical tissue-gel hybridization to minimize the loss of antigenicity while allowing permanent anchoring of biomolecules. We achieved success rates of 96% and 94% with synaptic antibodies for mouse and marmoset brains, respectively. Maximal preservation of antigenicity allows imaging of nanoscopic architectures in 1000-fold expanded tissues without additional signal amplification. eMAP-processed tissue gel can endure repeated staining and destaining without epitope loss or structural damage, enabling highly multiplexed proteomic analysis. We demonstrated the utility of eMAP as a nanoscopic proteomic interrogation tool by investigating molecular heterogeneity in inhibitory synapses in the mouse brain neocortex and characterizing the spatial distributions of synaptic proteins within synapses in mouse and marmoset brains.
ABSTRACT
OBJECTIVES: This study aimed to explore the relationship between online game addiction and aggression, self-control, and narcissistic personality traits, which are known as the psychological characteristics linked to "at-risk" populations for online game addiction. METHOD: A total of 1471 online game users (males 82.7%, females 17.3%, mean age 21.30+/-4.96) participated in this study and were asked to complete several self-report measures using an online response method. Questionnaires included demographic information and game use-related characteristics of the samples, the online game addiction scale (modified from Young's Internet addiction scale), the Buss-Perry aggression questionnaire, a self-control scale, and the narcissistic personality disorder scale. RESULTS: Our results indicated that aggression and narcissistic personality traits are positively correlated with online game addiction, whereas self-control is negatively correlated with online game addiction (p<0.001). In addition, a multiple regression analysis revealed that the extent of online game addiction could be predicted based on the person's narcissistic personality traits, aggression, self-control, interpersonal relationship, and occupation. However, only 20% of the variance in behavioral consequences was explained with the model. CONCLUSION: An interesting profile has emerged from the results of this study, suggesting that certain psychological characteristics such as aggression, self-control, and narcissistic personality traits may predispose some individuals to become addicted to online games. This result will deepen our understanding of the "at-risk" population for online game addiction and provide basic information that can contribute to developing a prevention program for people who are addicted to online games.
Subject(s)
Aggression/psychology , Behavior, Addictive/diagnosis , Internal-External Control , Internet , Personality Disorders/diagnosis , Video Games , Adolescent , Adult , Behavior, Addictive/epidemiology , Behavior, Addictive/psychology , Cross-Sectional Studies , Female , Health Surveys , Humans , Korea , Male , Personality Disorders/epidemiology , Personality Disorders/psychology , Surveys and QuestionnairesABSTRACT
Synaptic connections between hippocampal mossy fibers (MFs) and CA3 pyramidal neurons are essential for contextual memory encoding, but the molecular mechanisms regulating MF-CA3 synapses during memory formation and the exact nature of this regulation are poorly understood. Here we report that the activity-dependent transcription factor Npas4 selectively regulates the structure and strength of MF-CA3 synapses by restricting the number of their functional synaptic contacts without affecting the other synaptic inputs onto CA3 pyramidal neurons. Using an activity-dependent reporter, we identified CA3 pyramidal cells that were activated by contextual learning and found that MF inputs on these cells were selectively strengthened. Deletion of Npas4 prevented both contextual memory formation and this learning-induced synaptic modification. We further show that Npas4 regulates MF-CA3 synapses by controlling the expression of the polo-like kinase Plk2. Thus, Npas4 is a critical regulator of experience-dependent, structural, and functional plasticity at MF-CA3 synapses during contextual memory formation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , CA3 Region, Hippocampal/physiology , Memory/physiology , Mossy Fibers, Hippocampal/physiology , Neuronal Plasticity/physiology , Synapses/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/analysis , CA3 Region, Hippocampal/chemistry , Excitatory Postsynaptic Potentials/physiology , Female , Inhibitory Postsynaptic Potentials/physiology , Learning/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mossy Fibers, Hippocampal/chemistry , Synapses/chemistryABSTRACT
Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.
ABSTRACT
Indocyanine green (ICG) fluorescence imaging has been clinically used for noninvasive visualizations of vascular structures. We have previously developed a diagnostic system based on dynamic ICG fluorescence imaging for sensitive detection of vascular disorders. However, because high-dimensional raw data were used, the analysis of the ICG dynamics proved difficult. We used principal component analysis (PCA) in this study to extract important elements without significant loss of information. We examined ICG spatiotemporal profiles and identified critical features related to vascular disorders. PCA time courses of the first three components showed a distinct pattern in diabetic patients. Among the major components, the second principal component (PC2) represented arterial-like features. The explained variance of PC2 in diabetic patients was significantly lower than in normal controls. To visualize the spatial pattern of PCs, pixels were mapped with red, green, and blue channels. The PC2 score showed an inverse pattern between normal controls and diabetic patients. We propose that PC2 can be used as a representative bioimaging marker for the screening of vascular diseases. It may also be useful in simple extractions of arterial-like features.
Subject(s)
Diabetic Foot/diagnostic imaging , Image Interpretation, Computer-Assisted/methods , Optical Imaging/methods , Female , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacokinetics , Humans , Indocyanine Green/chemistry , Indocyanine Green/pharmacokinetics , Male , Middle Aged , Principal Component AnalysisABSTRACT
The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.
Subject(s)
Brain/metabolism , Imaging, Three-Dimensional/methods , Molecular Imaging/methods , Proteome/metabolism , Synapses/metabolism , Synapses/ultrastructure , Animals , Brain/ultrastructure , Female , Gene Expression Profiling/methods , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Immunoassay/methods , Male , Mice , Nerve Tissue Proteins/metabolism , Proteome/ultrastructure , Tissue DistributionABSTRACT
The smooth muscle cell is the principal component responsible for involuntary control of visceral organs, including vascular tonicity, secretion, and sphincter regulation. It is known that the neurotransmitters released from nerve endings increase the intracellular Ca(2+) level in smooth muscle cells followed by muscle contraction. We herein report that femtosecond laser pulses focused on the diffraction-limited volume can induce intracellular Ca(2+) increases in the irradiated smooth muscle cell without neurotransmitters, and locally increased intracellular Ca(2+) levels are amplified by calcium-induced calcium-releasing mechanisms through the ryanodine receptor, a Ca(2+) channel of the endoplasmic reticulum. The laser-induced Ca(2+) increases propagate to adjacent cells through gap junctions. Thus, ultrashort-pulsed lasers can induce smooth muscle contraction by controlling Ca(2+), even with optical stimulation of the diffraction-limited volume. This optical method, which leads to reversible and reproducible muscle contraction, can be used in research into muscle dynamics, neuromuscular disease treatment, and nanorobot control.
Subject(s)
Lasers , Muscle Contraction , Myocytes, Smooth Muscle/cytology , Optical Phenomena , Calcium/metabolism , Calcium Signaling , Extracellular Space/metabolism , Humans , Intracellular Space/metabolism , Myocytes, Smooth Muscle/metabolism , Time Factors , Urinary Bladder/cytology , Urinary Bladder/physiologyABSTRACT
Stroke is one of the common causes of death and disability. Despite extensive efforts in stroke research, therapeutic options for improving the functional recovery remain limited in clinical practice. Experimental stroke models using genetically modified mice could aid in unraveling the complex pathophysiology triggered by ischemic brain injury. Here, we optimized the procedure for generating mouse stroke model using an intraluminal suture in the middle cerebral artery and verified the blockage of blood flow using indocyanine green coupled with near infra-red radiation. The first week after the ischemic injury was critical for survivability. The survival rate of 11% in mice without any treatment but increased to 60% on administering prophylactic antibiotics. During this period, mice showed severe functional impairment but recovered spontaneously starting from the second week onward. Among the various behavioral tests, the pole tests and neurological severity score tests remained reliable up to 4 weeks after ischemia, whereas the rotarod and corner tests became less sensitive for assessing the severity of ischemic injury with time. Further, loss of body weight was also observed for up 4 weeks after ischemia induction. In conclusion, we have developed an improved approach which allows us to investigate the role of the cell death-related genes in the disease progression using genetically modified mice and to evaluate the modes of action of candidate drugs.
ABSTRACT
We engineered phototherapeutic sub-12 nm-sized polymeric micelles to treat malignant brain tumours (MBTs). The engineered nanoparticles in MBT cells enhanced the photocytotoxic efficiency more than 2.5-fold compared with parental and PEGylated photosensitisers (PSs). Increased subcellular co-localisation of PSs in mitochondria was observed.