Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish ( Danio rerio).

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer ( QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish ( Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.

Brain topology improved spiking neural network for efficient reinforcement learning of continuous control.

The brain topology highly reflects the complex cognitive functions of the biological brain after million-years of evolution. Learning from these biological topologies is a smarter and easier way to achieve brain-like intelligence with features of efficiency, robustness, and flexibility. Here we proposed a brain topology-improved spiking neural network (BT-SNN) for efficient reinforcement learning. First, hundreds of biological topologies are generated and selected as subsets of the Allen mouse brain topology with the help of the Tanimoto hierarchical clustering algorithm, which has been widely used in analyzing key features of the brain connectome. Second, a few biological constraints are used to filter out three key topology candidates, including but not limited to the proportion of node functions (e.g., sensation, memory, and motor types) and network sparsity. Third, the network topology is integrated with the hybrid numerical solver-improved leaky-integrated and fire neurons. Fourth, the algorithm is then tuned with an evolutionary algorithm named adaptive random search instead of backpropagation to guide synaptic modifications without affecting raw key features of the topology. Fifth, under the test of four animal-survival-like RL tasks (i.e., dynamic controlling in Mujoco), the BT-SNN can achieve higher scores than not only counterpart SNN using random topology but also some classical ANNs (i.e., long-short-term memory and multi-layer perception). This result indicates that the research effort of incorporating biological topology and evolutionary learning rules has much in store for the future.

Real-time analysis of large-scale neuronal imaging enables closed-loop investigation of neural dynamics.

Large-scale imaging of neuronal activities is crucial for understanding brain functions. However, it is challenging to analyze large-scale imaging data in real time, preventing closed-loop investigation of neural circuitry. Here we develop a real-time analysis system with a field programmable gate array-graphics processing unit design for an up to 500-megabyte-per-second image stream. Adapted to whole-brain imaging of awake larval zebrafish, the system timely extracts activity from up to 100,000 neurons and enables closed-loop perturbations of neural dynamics.

Piezo1-dependent regulation of pericyte proliferation by blood flow during brain vascular development.

Blood flow is known to regulate cerebrovascular development through acting on vascular endothelial cells (ECs). As an indispensable component of the neurovascular unit, brain pericytes physically couple with ECs and play vital roles in blood-brain barrier integrity maintenance and neurovascular coupling. However, it remains unclear whether blood flow affects brain pericyte development. Using in vivo time-lapse imaging of larval zebrafish, we monitored the developmental dynamics of brain pericytes and found that they proliferate to expand their population and increase their coverage to brain vessels. In combination with pharmacological and genetic approaches, we demonstrated that blood flow enhances brain pericyte proliferation through Piezo1 expressed in ECs. Moreover, we identified that EC-intrinsic Notch signaling is downstream of Piezo1 to promote the activation of Notch signaling in pericytes. Thus, our findings reveal a role of blood flow in pericyte proliferation, extending the functional spectrum of hemodynamics on cerebrovascular development.

Cascade Catalytic Nanoparticles Selectively Alkalize Cancerous Lysosomes to Suppress Cancer Progression and Metastasis.

Lysosomes are critical in modulating the progression and metastasis for various cancers. There is currently an unmet need for lysosomal alkalizers that can selectively and safely alter the pH and inhibit the function of cancer lysosomes. Here an effective, selective, and safe lysosomal alkalizer is reported that can inhibit autophagy and suppress tumors in mice. The lysosomal alkalizer consists of an iron oxide core that generates hydroxyl radicals (•OH) in the presence of excessive H+ and hydrogen peroxide inside cancer lysosomes and cerium oxide satellites that capture and convert •OH into hydroxide ions. Alkalized lysosomes, which display impaired enzyme activity and autophagy, lead to cancer cell apoptosis. It is shown that the alkalizer effectively inhibits both local and systemic tumor growth and metastasis in mice. This work demonstrates that the intrinsic properties of nanoparticles can be harnessed to build effective lysosomal alkalizers that are both selective and safe.

Circadian regulation of developmental synaptogenesis via the hypocretinergic system.

The circadian clock orchestrates a wide variety of physiological and behavioral processes, enabling animals to adapt to daily environmental changes, particularly the day-night cycle. However, the circadian clock's role in the developmental processes remains unclear. Here, we employ the in vivo long-term time-lapse imaging of retinotectal synapses in the optic tectum of larval zebrafish and reveal that synaptogenesis, a fundamental developmental process for neural circuit formation, exhibits circadian rhythm. This rhythmicity arises primarily from the synapse formation rather than elimination and requires the hypocretinergic neural system. Disruption of this synaptogenic rhythm, by impairing either the circadian clock or the hypocretinergic system, affects the arrangement of the retinotectal synapses on axon arbors and the refinement of the postsynaptic tectal neuron's receptive field. Thus, our findings demonstrate that the developmental synaptogenesis is under hypocretin-dependent circadian regulation, suggesting an important role of the circadian clock in neural development.

Structure and function in artificial, zebrafish and human neural networks.

Network science provides a set of tools for the characterization of the structure and functional behavior of complex systems. Yet a major problem is to quantify how the structural domain is related to the dynamical one. In other words, how the diversity of dynamical states of a system can be predicted from the static network structure? Or the reverse problem: starting from a set of signals derived from experimental recordings, how can one discover the network connections or the causal relations behind the observed dynamics? Despite the advances achieved over the last two decades, many challenges remain concerning the study of the structure-dynamics interplay of complex systems. In neuroscience, progress is typically constrained by the low spatio-temporal resolution of experiments and by the lack of a universal inferring framework for empirical systems. To address these issues, applications of network science and artificial intelligence to neural data have been rapidly growing. In this article, we review important recent applications of methods from those fields to the study of the interplay between structure and functional dynamics of human and zebrafish brain. We cover the selection of topological features for the characterization of brain networks, inference of functional connections, dynamical modeling, and close with applications to both the human and zebrafish brain. This review is intended to neuroscientists who want to become acquainted with techniques from network science, as well as to researchers from the latter field who are interested in exploring novel application scenarios in neuroscience.

Midazolam Exposure Impedes Oligodendrocyte Development via the Translocator Protein and Impairs Myelination in Larval Zebrafish.

Anesthetics are commonly used in various medical procedures. Accumulating evidence suggests that early-life anesthetics exposure in infants and children affects brain development, causing psychiatric and neurological disorders. However, the underlying mechanisms are poorly understood. Using zebrafish larvae as a model, we found that the proliferation and migration of oligodendrocyte progenitor cells (OPCs) were severely impaired by the exposure of midazolam (MDZ), an anesthetic widely used in pediatric surgery and intensive care medicine, leading to a reduction of oligodendroglial lineage cell in the dorsal spinal cord. This defect was mimicked by the bath application of translocator protein (TSPO) agonists and partially rescued by genetic downregulation of TSPO. Cell transplantation experiments showed that requirement of TSPO for MDZ-induced oligodendroglial lineage cell defects is cell-autonomous. Furthermore, transmission electron microscopy and in vivo electrophysiological recording experiments demonstrated that MDZ exposure caused axon hypomyelination and action potential propagation retardation, resulting in delayed behavior initiation. Thus, our findings reveal that MDZ affects oligodendroglial lineage cell development and myelination in young animals, raising the care about its clinic use in infants and children.

A sensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo.

The purinergic transmitter ATP (adenosine 5'-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. Here, we developed a sensitive GPCR activation-based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, has ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including stimuli-induced and spontaneous ATP release in primary hippocampal cultures, injury-induced ATP release in a zebrafish model, and lipopolysaccharides-induced ATP-release events in individual astrocytes in the mouse cortex. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.

The landscape of regulatory genes in brain-wide neuronal phenotypes of a vertebrate brain.

Multidimensional landscapes of regulatory genes in neuronal phenotypes at whole-brain levels in the vertebrate remain elusive. We generated single-cell transcriptomes of ~67,000 region- and neurotransmitter/neuromodulator-identifiable cells from larval zebrafish brains. Hierarchical clustering based on effector gene profiles ('terminal features') distinguished major brain cell types. Sister clusters at hierarchical termini displayed similar terminal features. It was further verified by a population-level statistical method. Intriguingly, glutamatergic/GABAergic sister clusters mostly expressed distinct transcription factor (TF) profiles ('convergent pattern'), whereas neuromodulator-type sister clusters predominantly expressed the same TF profiles ('matched pattern'). Interestingly, glutamatergic/GABAergic clusters with similar TF profiles could also display different terminal features ('divergent pattern'). It led us to identify a library of RNA-binding proteins that differentially marked divergent pair clusters, suggesting the post-transcriptional regulation of neuron diversification. Thus, our findings reveal multidimensional landscapes of transcriptional and post-transcriptional regulators in whole-brain neuronal phenotypes in the zebrafish brain.

The brain harbors an astounding diversity of interconnected cells. Each cell contains the same basic set of genetic instructions, but only a fraction of the genome is used in each cell to assemble proteins. This selective gene expression gives rise to each cell's characteristic properties, such as their shape and location, or whether they can activate or inhibit neighbouring cells. How these defining features are encoded on a genetic level and selectively activated in cells to produce such diversity in the brain is not fully understood. One way to study gene expression in single cells involves profiling the transcriptome, the full range of intermediary RNA molecules a cell produces from its genes to make proteins. Zhang et al. used transcriptome profiling to better understand how thousands of regulatory genes encoding regulatory proteins called transcription factors create different types of neurons in the zebrafish brain, which is similar to but much simpler than the human brain. To do so, they analysed transcriptome data extracted from cell populations located in specific brain regions and displaying different properties. Zhang et al. identified distinct clusters of neurons in the larval zebrafish brain. Mathematical models then analysed the transcriptome profiles of these neuronal clusters with characteristic features. They revealed that neurons with similar characteristics did not necessarily share the same transcription factors. In other words, distinct sets of transcription factors gave rise to the same types of cells. Zhang et al. described this observation as a 'convergent' pattern. On the contrary, some neurons with dissimilar features expressed the same sorts of transcription factors, suggesting a 'divergent' developmental pattern also exists. In summary, this work sheds light on variable gene expression patterns akin to design principles that shape neuronal diversity in the brain. It gives a new appreciation of how neuronal subtypes result from a complex set of regulatory factors controlling gene expression.

A protocol for simultaneous Ca2+ and morphology imaging of brain endothelial tip cells in larval zebrafish.

Endothelial tip cells (ETCs) located at growing blood vessels display high morphological dynamics and associated intracellular Ca2+ activities with different spatiotemporal patterns during migration. Examining the Ca2+ activity and morphological dynamics of ETCs will provide an insight for understanding the mechanism of vascular development in organs, including the brain. Here, we describe a method for simultaneous monitoring and relevant analysis of the Ca2+ activity and morphology of growing brain ETCs in larval zebrafish. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).

Imaging volumetric dynamics at high speed in mouse and zebrafish brain with confocal light field microscopy.

A detailed understanding of the function of neural networks and how they are supported by a dynamic vascular system requires fast three-dimensional imaging in thick tissues. Here we present confocal light field microscopy, a method that enables fast volumetric imaging in the brain at depths of hundreds of micrometers. It uses a generalized confocal detection scheme that selectively collects fluorescent signals from the in-focus volume and provides optical sectioning capability to improve imaging resolution and sensitivity in thick tissues. We demonstrate recording of whole-brain calcium transients in freely swimming zebrafish larvae and observe behaviorally correlated activities in single neurons during prey capture. Furthermore, in the mouse brain, we detect neural activities at depths of up to 370 µm and track blood cells at 70 Hz over a volume of diameter 800 µm × thickness 150 µm and depth of up to 600 µm.

H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish.

BACKGROUND: Hydrogen sulphide (H2S) is considered as the third member of the gasotransmitter family, along with nitric oxide (NO) and carbon monoxide. H2S has been reported to induce angiogenesis by promoting the growth, migration and tube-like structure formation of endothelial cells. Those studies were conducted in conditions of cell culture, mouse Matrigel plug assay model, rat wound healing model or rat hindlimb ischaemia model. Recent in vivo studies showed the physiological importance of H2S in muscle angiogenesis. However, the importance of endogenous H2S for brain angiogenesis during development remains unknown. We therefore aimed at determining the role of H2S in brain vascular development. METHODS AND RESULTS: Both knockdown and knockout of H2S-producing enzymes, cystathionine ß-synthase (cbs) and cystathionine γ-lyase (cth), using morpholino oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9-mediated mutation, impaired brain vascular development of larval zebrafish. Incubation with the slow-releasing H2S donor GYY4137 alleviated the defects of brain vascular development in cbs and cth morphants. Quantitative analysis of the midbrain vascular network showed that H2S enhances angiogenesis without affecting the topological structure of the brain vasculature. Mechanically, nitric oxide synthase 2a (nos2a) expression and NO production were decreased in both cbs and cth morphants. Overexpression of nos2a by coinjection of cbs or cth MO with full-length zebrafish nos2a mRNA alleviated the brain vascular developmental defects in cbs and cth morphants. CONCLUSION: We conclude that H2S promotes brain developmental angiogenesis via the NOS/NO pathway in zebrafish.

Piezo1-Mediated Ca2+ Activities Regulate Brain Vascular Pathfinding during Development.

During development, endothelial tip cells (ETCs) located at the leading edge of growing vascular plexus guide angiogenic sprouts to target vessels, and thus, ETC pathfinding is fundamental for vascular pattern formation in organs, including the brain. However, mechanisms of ETC pathfinding remain largely unknown. Here, we report that Piezo1-mediated Ca2+ activities at primary branches of ETCs regulate branch dynamics to accomplish ETC pathfinding during zebrafish brain vascular development. ETC branches display spontaneous local Ca2+ transients, and high- and low-frequency Ca2+ transients cause branch retraction through calpain and branch extension through nitric oxide synthase, respectively. These Ca2+ transients are mainly mediated by Ca2+-permeable Piezo1 channels, which can be activated by mechanical force, and mutating piezo1 largely impairs ETC pathfinding and brain vascular patterning. These findings reveal that Piezo1 and downstream Ca2+ signaling act as molecular bases for ETC pathfinding and highlight a novel function of Piezo1 and Ca2+ in vascular development.

A highly sensitive and selective nanosensor for near-infrared potassium imaging.

Potassium ion (K+) concentration fluctuates in various biological processes. A number of K+ probes have been developed to monitor such fluctuations through optical imaging. However, the currently available K+ probes are far from being sensitive enough in detecting physiological fluctuations in living animals. Furthermore, the monitoring of deep tissues is not applicable because of short-wavelength excitation prevailingly used so far. Here, we report a highly sensitive and selective nanosensor for near-infrared (NIR) K+ imaging in living cells and animals. The nanosensor is constructed by encapsulating upconversion nanoparticles (UCNPs) and a commercial K+ indicator in the hollow cavity of mesoporous silica nanoparticles, followed by coating a K+-selective filter membrane. The membrane adsorbs K+ from the medium and filters out interfering cations. The UCNPs convert NIR to ultraviolet light, which excites the K+ indicator, thus allowing the detection of the fluctuations of K+ concentration in cultured cells and intact mouse brains.

Near-Infrared Voltage Nanosensors Enable Real-Time Imaging of Neuronal Activities in Mice and Zebrafish.

Optical voltage sensors with the ability to monitor neuronal activities are invaluable tools for studying information processing of the brain. However, the current genetically encoded voltage indicators usually require high-power visible light for excitation and are limited to genetically addressable model animals. Here, we report a near-infrared (NIR)-excited nongenetic voltage nanosensor that achieves stable recording of neuronal membrane potential in intact animals. The nanosensor is composed of a Förster resonance energy transfer (FRET) pair, the outer membrane-anchored upconversion nanoparticle (UCNP), and the membrane-embedded dipicrylamine (DPA). The negative charge of DPA allows membrane potential fluctuation to affect the distance between the DPA and UCNP, therefore changing the FRET efficiency. Consequently, the emission intensity of the nanosensor can report the membrane potential. Using the nanosensor, we monitor not only electrically evoked changes in the membrane potential of cultured cells but also sensory responses of neurons in intact zebrafish and brain state-modulated subthreshold activities of cortical neurons in intact mice.