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.

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.

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.

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).

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.

One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting.

The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.

Müller Glial Cells Participate in Retinal Waves via Glutamate Transporters and AMPA Receptors.

Retinal waves, the spontaneous patterned neural activities propagating among developing retinal ganglion cells (RGCs), instruct the activity-dependent refinement of visuotopic maps. Although it is known that the wave is initiated successively by amacrine cells and bipolar cells, the behavior and function of glia in retinal waves remain unclear. Using multiple in vivo methods in larval zebrafish, we found that Müller glial cells (MGCs) display wave-like spontaneous activities, which start at MGC processes within the inner plexiform layer, vertically spread to their somata and endfeet, and horizontally propagate into neighboring MGCs. MGC waves depend on glutamatergic signaling derived from bipolar cells. Moreover, MGCs express both glia-specific glutamate transporters and the AMPA subtype of glutamate receptors. The AMPA receptors mediate MGC calcium activities during retinal waves, whereas the glutamate transporters modulate the occurrence of retinal waves. Thus, MGCs can sense and regulate retinal waves via AMPA receptors and glutamate transporters, respectively.

Role of Olfactorily Responsive Neurons in the Right Dorsal Habenula-Ventral Interpeduncular Nucleus Pathway in Food-Seeking Behaviors of Larval Zebrafish.

The habenula (Hb) plays important roles in emotion-related behaviors. Besides receiving inputs from the limbic system and basal ganglia, Hb also gets inputs from multiple sensory modalities. Sensory responses of Hb neurons in zebrafish are asymmetrical: the left dorsal Hb and right dorsal Hb (dHb) preferentially respond to visual and olfactory stimuli, respectively, implying different functions of the left and right dHb. While visual responses of the left dHb (L-dHb) have been implicated in light-preference behavior, the significance of olfactory responses of the right dHb (R-dHb) remains under-examined. It was reported that the R-dHb can gate innate attraction to a bile salt. However, considering a broad range of odors that R-dHb respond to, it is of interest to examine the role of R-dHb in other olfactory behaviors, especially food seeking, which is essential for animals' survival. Here, using in vivo whole-cell recording and calcium imaging, we first characterized food extract-evoked responses of Hb neurons. Responsive neurons preferentially locate in the R- but not L-dHb and exhibit either ON- (~87%) or OFF-type responses (~13%). Interestingly, this right-to-left asymmetry of olfactory responses converts into a ventral-to-dorsal pattern in the interpeduncular nucleus (IPN), a main downstream target of Hb. Combining behavior assay, we further found that genetic dysfunction or lesion of the R-dHb and its corresponding downstream ventral IPN (V-IPN) impair the food seeking-associated increase of swimming activity. Thus, our study indicates that the asymmetrical olfactory response in the R-dHb to V-IPN pathway plays an important role in food-seeking behavior of zebrafish larvae.

The Locus Coeruleus Modulates Intravenous General Anesthesia of Zebrafish via a Cooperative Mechanism.

How general anesthesia causes loss of consciousness has been a mystery for decades. It is generally thought that arousal-related brain nuclei, including the locus coeruleus (LC), are involved. Here, by monitoring locomotion behaviors and neural activities, we developed a larval zebrafish model for studying general anesthesia induced by propofol and etomidate, two commonly used intravenous anesthetics. Local lesion of LC neurons via two-photon laser-based ablation or genetic depletion of norepinephrine (NE; a neuromodulator released by LC neurons) via CRISPR/Cas9-based mutation of dopamine-ß-hydroxylase (dbh) accelerates induction into and retards emergence from general anesthesia. Mechanistically, in vivo whole-cell recording revealed that both anesthetics suppress LC neurons' activity through a cooperative mechanism, inhibiting presynaptic excitatory inputs and inducing GABAA receptor-mediated hyperpolarization of these neurons. Thus, our study indicates that the LC-NE system plays a modulatory role in both induction of and emergence from intravenous general anesthesia.

A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis.

The retinotectal synapse in larval zebrafish, combined with live time-lapse imaging, provides an advantageous model for study of the development and remodelling of central synapses in vivo. In previous studies, these synapses were labelled by transient expression of fluorescence-tagged synaptic proteins, which resulted in the dramatic variation of labelling patterns in each larva. Here, using GAL4-Upstream Activating Sequence (GAL4-UAS) methodology, we generated stable transgenic lines, which express EGFP-tagged synaptophysin (a presynaptic protein) in retinal ganglion cells (RGCs), to reliably label the pre-synaptic site of retinotectal synapses. This tool avoids the variable labelling of RGCs that occurs in transient transgenic larvae. We obtained several stable transgenic lines that differ consistently in the number of labelled RGCs. Using stable lines that consistently had a single labelled RGC, we could trace synaptogenic dynamics on an individual RGC axonal arbor across different developmental stages. In the stable lines that consistently had multiple labelled RGCs, we could simultaneously monitor both pre- and post-synaptic compartments by combining transient labelling of post-synaptic sites on individual tectal neurons. These tools allowed us to investigate molecular events underlying synaptogenesis and found that the microRNA-132 (miR-132) is required for developmental synaptogenesis. Thus, these transgenic zebrafish stable lines provide appropriate tools for studying central synaptogenesis and underlying molecular mechanisms in intact vertebrate brain.

All-optical imaging and manipulation of whole-brain neuronal activities in behaving larval zebrafish.

All-optical interrogation of population neuron activity is a promising approach to deciphering the neural circuit mechanisms supporting brain functions. However, this interrogation is currently limited to local brain areas. Here, we incorporate patterned photo-stimulation into light-sheet microscopy, allowing simultaneous targeted optogenetic manipulation and brain-wide monitoring of the neuronal activities of head-restrained behaving larval zebrafish. Using this system, we photo-stimulate arbitrarily selected neurons (regions as small as ~10-20 neurons in 3D) in zebrafish larvae with pan-neuronal expression of a spectrally separated calcium indicator, GCaMP6f, and an activity actuator, ChrimsonR, and observe downstream neural circuit activation and behavior generation. This approach allows us to dissect the causal role of neural circuits in brain functions and behavior generation.

[The structure and function of habenula].

The habenula (Hb) is an evolutionarily conserved diencephalic structure in vertebrates. It is considered as an emotion center and plays critical roles in regulating diverse types of emotion-related behaviors, including anxiety, fear, reward, depression, and nicotine withdrawal. On the one hand, action selection- and emotion-relevant inputs are transferred to the Hb through the basal ganglia and limbic system, respectively. At the same time, sensory inputs of multiple modalities also converge on the Hb. Among them, the visual input of the Hb from the retina ganglion cells ‒ thalamus pathway has been found to play a critical role in light-preference behavior of zebrafish. On the other hand, the Hb projects to two main neuromodulatory systems, the dopaminergic system and the serotoninergic system. As the Hb receives both internal emotion inputs and external sensory inputs and regulates the function of neuromodulatory systems, its functions are quite diverse and complex. In this review, we summarize the progress in both the structure and connection of the Hb and propose future study direction.

General anesthetics protects against cardiac arrest-induced brain injury by inhibiting calcium wave propagation in zebrafish.

Cardiac arrest is a leading cause of death and disability worldwide. Although many victims are initially resuscitated, they often suffer from serious brain injury, even leading to a "persistent vegetative state". Therefore, it is need to explore therapies which restore and protect brain function after cardiac arrest. In the present study, using Tg (HuC:GCaMP5) zebrafish as a model, we found the zebrafish brain generated a burst of Ca2+ wave after cardiac arrest by in vivo time-lapse confocal imaging. The Ca2+ wave was firstly initiated at hindbrain and then sequentially propagated to midbrain and telencephalon, the neuron displayed Ca2+ overload after Ca2+ wave propagation. Consistent with this, our study further demonstrated neuronal apoptosis was increased in cardiac arrest zebrafish by TUNEL staining. The cardiac arrest-induced Ca2+ wave propagation can be prevented by general anesthetics such as midazolam or ketamine pretreatment. Moreover, midazolam or ketamine pretreatment dramatically decreased the neuronal apoptosis and improved the survival rate in CA zebrafish. Taken together, these findings provide the first in vivo evidence that general anesthetics pretreatment protects against cardiac arrest-induced brain injury by inhibiting calcium wave propagation in zebrafish.

Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity.

Vascular integrity helps maintain brain microenvironment homeostasis, which is critical for the normal development and function of the central nervous system. It is known that neural cells can regulate brain vascular integrity. However, due to the high complexity of neurovascular interactions involved, understanding of the neural regulation of brain vascular integrity is still rudimentary. Using intact zebrafish larvae and cultured rodent brain cells, we find that neurons transfer miR-132, a highly conserved and neuron-enriched microRNA, via secreting exosomes to endothelial cells (ECs) to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase (eef2k). Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity. Our study indicates that miR-132 acts as an intercellular signal mediating neural regulation of the brain vascular integrity and suggests that the neuronal exosome is a novel avenue for neurovascular communication.

Left Habenula Mediates Light-Preference Behavior in Zebrafish via an Asymmetrical Visual Pathway.

Habenula (Hb) plays critical roles in emotion-related behaviors through integrating inputs mainly from the limbic system and basal ganglia. However, Hb also receives inputs from multiple sensory modalities. The function and underlying neural circuit of Hb sensory inputs remain unknown. Using larval zebrafish, we found that left dorsal Hb (dHb, a homolog of mammalian medial Hb) mediates light-preference behavior by receiving visual inputs from a specific subset of retinal ganglion cells (RGCs) through eminentia thalami (EmT). Loss- and gain-of-function manipulations showed that left, but not right, dHb activities, which encode environmental illuminance, are necessary and sufficient for light-preference behavior. At circuit level, left dHb neurons receive excitatory monosynaptic inputs from bilateral EmT, and EmT neurons are contacted mainly by sustained ON-type RGCs at the arborization field 4 of retinorecipient brain areas. Our findings discover a previously unidentified asymmetrical visual pathway to left Hb and its function in mediating light-preference behavior. VIDEO ABSTRACT.

China Brain Project: Basic Neuroscience, Brain Diseases, and Brain-Inspired Computing.

The China Brain Project covers both basic research on neural mechanisms underlying cognition and translational research for the diagnosis and intervention of brain diseases as well as for brain-inspired intelligence technology. We discuss some emerging themes, with emphasis on unique aspects.

Stereotyped initiation of retinal waves by bipolar cells via presynaptic NMDA autoreceptors.

Glutamatergic retinal waves, the spontaneous patterned neural activities propagating among developing retinal ganglion cells (RGCs), instruct the activity-dependent refinement of visuotopic maps. However, its initiation and underlying mechanism remain largely elusive. Here using larval zebrafish and multiple in vivo approaches, we discover that bipolar cells (BCs) are responsible for the generation of glutamatergic retinal waves. The wave originates from BC axon terminals (ATs) and propagates laterally to nearby BCs and vertically to downstream RGCs and the optic tectum. Its initiation is triggered by the activation of and consequent glutamate release from BC ATs, and is mediated by the N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) expressed at these ATs. Intercellular asymmetry of NMDAR expression at BC ATs enables the preferential initiation of waves at the temporal retina, where BC ATs express more NMDARs. Thus, our findings indicate that glutamatergic retinal waves are initiated by BCs through a presynaptic NMDA autoreceptor-dependent process.