Zebrafish Spinal Cord Research Articles

Fast and light-efficient remote focusing for volumetric voltage imaging

Voltage imaging holds great potential for biomedical research by enabling noninvasive recording of the electrical activity of excitable cells such as neurons or cardiomyocytes. Camera-based detection can record from hundreds of cells in parallel, but imaging entire volumes is limited by the need to focus through the sample at high speeds. Remote focusing techniques can remedy this drawback, but have so far been either too slow or light-inefficient. Here, we introduce flipped image remote focusing, a remote focusing method that doubles the light efficiency compared to conventional beamsplitter-based techniques and enables high-speed volumetric voltage imaging at 500 volumes/s. We show the potential of our approach by combining it with light sheet imaging in the zebrafish spinal cord to record from >100 spontaneously active neurons in parallel.

Il34 rescues metronidazole-induced impairment of spinal cord regeneration in zebrafish central nervous system.

Metronidazole (MTZ), a commonly used anti-infective drug in clinical practice, has also been employed as a prodrug in cell-targeted ablation systems in scientific research, exhibiting significant application value. However, it has been demonstrated that MTZ can induce neurotoxic symptoms to some extent during its use, and there is currently a lack of effective means to circumvent its toxicity in both clinical and research settings, which limits its application. Therefore, exploring the specific mechanisms underlying MTZ-induced neurotoxic symptoms and elucidating countermeasures will enhance the practical value of MTZ. In this study, using a zebrafish spinal cord injury regeneration model, we confirmed that MTZ neurotoxicity leads to impaired axon regeneration in the central nervous system. By overexpressing il34 in the central nervous system of zebrafish, we eliminated the inhibitory effect of MTZ on axonal regeneration and demonstrated that the pro-regenerative effect against MTZ neurotoxicity is not caused by excessive macrophages/microglia chemoattracted by interleukin 34(Il34). Transcriptome sequencing analysis and GO enrichment analysis of differentially expressed genes between groups revealed that Il34 may counteract MTZ neurotoxicity and promote spinal cord injury repair through biological processes that enhance cellular adhesion and cell location. In summary, our work uncovers a possible cause of MTZ neurotoxicity and provides a new perspective for eliminating MTZ toxicity.

Transcriptional regulators with broad expression in the zebrafish spinal cord.

The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.

Neuroprotective gap-junction-mediated bystander transformations in the adult zebrafish spinal cord after injury

The adult zebrafish spinal cord displays an impressive innate ability to regenerate after traumatic insults, yet the underlying adaptive cellular mechanisms remain elusive. Here, we show that while the cellular and tissue responses after injury are largely conserved among vertebrates, the large-size fast spinal zebrafish motoneurons are remarkably resilient by remaining viable and functional. We also reveal the dynamic changes in motoneuron glutamatergic input, excitability, and calcium signaling, and we underscore the critical role of calretinin (CR) in binding and buffering the intracellular calcium after injury. Importantly, we demonstrate the presence and the dynamics of a neuron-to-neuron bystander neuroprotective biochemical cooperation mediated through gap junction channels. Our findings support a model in which the intimate and dynamic interplay between glutamate signaling, calcium buffering, gap junction channels, and intercellular cooperation upholds cell survival and promotes the initiation of regeneration.

P2X7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury.

In contrast to mammals, zebrafish undergo successful neural regeneration following spinal cord injury. Spinal cord ependymo-radial glia (ERG) undergo injury-induced proliferation and neuronal differentiation to replace damaged cells and restore motor function. However, the molecular cues driving these processes remain elusive. Here, we demonstrate that the evolutionarily conserved P2X7 receptors are widely distributed on neurons and ERG within the zebrafish spinal cord. At the protein level, the P2X7 receptor expressed in zebrafish is a truncated splice variant of the full-length variant found in mammals. The protein expression of this 50 kDa isoform was significantly downregulated at 7 days post-injury (dpi) but returned to basal levels at 14 dpi when compared to naïve controls. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi but did not affect neuronal differentiation at 14 dpi. Our findings suggest that unlike in mammals, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish and may also work to curb the proliferative response of ERG following injury.

Microtubule polarity determines the lineage of embryonic neural precursor in zebrafish spinal cord.

The phenomenal diversity of neuronal types in the central nervous system is achieved in part by the asymmetric division of neural precursors. In zebrafish neural precursors, asymmetric dispatch of Sara endosomes (with its Notch signaling cargo) functions as fate determinant which mediates asymmetric division. Here, we found two distinct pools of neural precursors based on Sara endosome inheritance and spindle-microtubule enrichment.Symmetric or asymmetric levels of spindle-microtubules drivedifferently Sara endosomes inheritance and predict neural precursor lineage. We uncover that CAMSAP2a/CAMSAP3a and KIF16Ba govern microtubule asymmetry and endosome motility, unveiling the heterogeneity of neural precursors.Using a plethora of physical and cell biological assays, we determined thephysical parameters andmolecularmechanismsbehind microtubule asymmetries and biased endosome motility. Evolutionarily, the values of those parameters explain why all sensory organ precursor cells are asymmetric in flies while, in zebrafish spinal cord, two populations ofneural precursors (symmetric vs asymmetric) are possible.

Synaptic input and Ca2+ activity in zebrafish oligodendrocyte precursor cells contribute to myelin sheath formation.

In the nervous system, only one type of neuron-glial synapse is known to exist: that between neurons and oligodendrocyte precursor cells (OPCs), yet their composition, assembly, downstream signaling and in vivo functions remain largely unclear. Here, we address these questions using in vivo microscopy in zebrafish spinal cord and identify postsynaptic molecules PSD-95 and gephyrin in OPCs. The puncta containing these molecules in OPCs increase during early development and decrease upon OPC differentiation. These puncta are highly dynamic and frequently assemble at 'hotspots'. Gephyrin hotspots and synapse-associated Ca2+ activity in OPCs predict where a subset of myelin sheaths forms in differentiated oligodendrocytes. Further analyses reveal that spontaneous synaptic release is integral to OPC Ca2+ activity, while evoked synaptic release contributes only in early development. Finally, disruption of the synaptic genes dlg4a/dlg4b, gphnb and nlgn3b impairs OPC differentiation and myelination. Together, we propose that neuron-OPC synapses are dynamically assembled and can predetermine myelination patterns through Ca2+ signaling.

Controlling the Immune Response to Zebrafish Spinal Cord Injury via Extracellular Vesicles Secreted by Activated Monocyte-like Cells

Controlling the Immune Response to Zebrafish Spinal Cord Injury via Extracellular Vesicles Secreted by Activated Monocyte-like Cells

In toto imaging of glial JNK signaling during larval zebrafish spinal cord regeneration.

Identification of signaling events that contribute to innate spinal cord regeneration in zebrafish can uncover new targets for modulating injury responses of the mammalian central nervous system. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, dynamic and regional regulation of JNK signaling help to direct glial cell behaviors during innate spinal cord regeneration.

Mechanical Ablation of Larval Zebrafish Spinal Cord.

Unlike mammals, adult and larval zebrafish exhibit robust regeneration following traumatic spinal cord injury. This remarkable regenerative capacity, combined with exquisite imaging capabilities and an abundance of powerful genetic techniques, has established the zebrafish as an important vertebrate model for the study of neural regeneration. Here, we describe a protocol for the complete mechanical ablation of the larval zebrafish spinal cord. With practice, this protocol can be used to reproducibly injure upward of 100 samples per hour, facilitating the high-throughput screening of factors involved in spinal cord regeneration and repair.

New insights into zebrafish spinal cord repair.

New insights into zebrafish spinal cord repair.

Development and repair of blood vessels in the zebrafish spinal cord.

The vascular system is inefficiently repaired after spinal cord injury (SCI) in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord (SC) and restore motility, but the vascular response to injury has not been investigated. Here, we describe the zebrafish SC blood vasculature, starting in development with the initial vessel ingression in a body size-dependent manner, the acquisition of perivascular support and the establishment of ventral to dorsal blood circulation. The vascular organization grows in complexity and displays multiple barrier specializations in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and axons. Vascular repair involves an early burst of angiogenesis that creates dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-SC barrier is re-established. This study demonstrates that zebrafish can successfully re-vascularize the spinal tissue, reinforcing the value of this organism as a regenerative model for SCI.

Sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons

Sox transcription factors are crucial for vertebrate nervous system development. In zebrafish embryo, sox1 genes are expressed in neural progenitor cells and neurons of ventral spinal cord. Our recent study revealed that the loss of sox1a and sox1b function results in a significant decline of V2 subtype neurons (V2s). Using single-cell RNA sequencing, we analyzed the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four time points during embryonic development, employing the Tg(sox1a:eGFP) line. In addition to previously characterized sox1a-expressing neurons, we discovered the expression of sox1a in late-developing intraspinal serotonergic neurons (ISNs). Developmental trajectory analysis suggests that ISNs arise from lateral floor plate (LFP) progenitor cells. Pharmacological inhibition of the Notch signaling pathway revealed its role in negatively regulating LFP progenitor cell differentiation into ISNs. Our findings highlight the zebrafish LFP as a progenitor domain for ISNs, alongside known Kolmer-Agduhr (KA) and V3 interneurons.

Transcription Pattern of Neurotrophic Factors and Their Receptors in Adult Zebrafish Spinal Cord.

In vertebrates, neurotrophins and their receptors play a fundamental role in the central and peripheral nervous systems. Several studies reported that each neurotrophin/receptor signalling pathway can perform various functions during axon development, neuronal growth, and plasticity. Previous investigations in some fish species have identified neurotrophins and their receptors in the spinal cord under physiological conditions and after injuries, highlighting their potential role during regeneration. In our study, for the first time, we used an excellent animal model, the zebrafish (Danio rerio), to compare the mRNA localization patterns of neurotrophins and receptors in the spinal cord. We quantified the levels of mRNA using qPCR, and identified the transcription pattern of each neurotrophin/receptor pathway via in situ hybridization. Our data show that ngf/trka are the most transcribed members in the adult zebrafish spinal cord.

Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate.

Oligodendrocytes in the central nervous system exhibit significant variability in the number of myelin sheaths that are supported by each cell, ranging from 1 to 50 (1-8). Myelin production during development is dynamic and involves both sheath formation and loss (3, 9-13). However, how these parameters are balanced to generate this heterogeneity in sheath number has not been thoroughly investigated. To explore this question, we combined extensive time-lapse and longitudinal imaging of oligodendrocytes in the developing zebrafish spinal cord to quantify sheath initiation and loss. Surprisingly, we found that oligodendrocytes repetitively ensheathed the same axons multiple times before any stable sheaths were formed. Importantly, this repetitive ensheathment was independent of neuronal activity. At the level of individual oligodendrocytes, each cell initiated a highly variable number of total ensheathments. However, ~80-90% of these ensheathments always disappeared, an unexpectedly high, but consistent rate of loss. The dynamics of this process indicated rapid membrane turnover as ensheathments were formed and lost repetitively on each axon. To better understand how these sheath initiation dynamics contribute to sheath accumulation and stabilization, we disrupted membrane recycling by expressing a dominant-negative mutant form of Rab5. Oligodendrocytes over-expressing this mutant did not show a change in early sheath initiation dynamics but did lose a higher percentage of ensheathments in the later stabilization phase. Overall, oligodendrocyte sheath number is heterogeneous because each cell repetitively initiates a variable number of total ensheathments that are resolved through a consistent stabilization rate.

SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice.

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.

Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

Intersegmental coordination of the central pattern generator via interleaved electrical and chemical synapses in zebrafish spinal cord.

A significant component of the repetitive dynamics during locomotion in vertebrates is generated within the spinal cord. The legged locomotion of mammals is most likely controled by a hierarchical, multi-layer spinal network structure, while the axial circuitry generating the undulatory swimming motion of animals like lamprey is thought to have only a single layer in each segment. Recent experiments have suggested a hybrid network structure in zebrafish larvae in which two types of excitatory interneurons (V2a-I and V2a-II) both make first-order connections to the brain and last-order connections to the motor pool. These neurons are connected by electrical and chemical synapses across segments. Through computational modeling and an asymptotic perturbation approach we show that this interleaved interaction between the two neuron populations allows the spinal network to quickly establish the correct activation sequence of the segments when starting from random initial conditions, as needed for a swimming spurt, and to reduce the dependence of the intersegmental phase difference (ISPD) of the oscillations on the swimming frequency. The latter reduces the frequency dependence of the waveform of the swimming motion. In the model the reduced frequency dependence is largely due to the different impact of chemical and electrical synapses on the ISPD and to the significant spike-frequency adaptation that has been observed experimentally in V2a-II neurons, but not in V2a-I neurons. Our model makes experimentally testable predictions and points to a benefit of the hybrid structure for undulatory locomotion that may not be relevant for legged locomotion.

Mixed synapses reconcile violations of the size principle in zebrafish spinal cord.

Mixed electrical-chemical synapses potentially complicate electrophysiological interpretations of neuronal excitability and connectivity. Here, we disentangle the impact of mixed synapses within the spinal locomotor circuitry of larval zebrafish. We demonstrate that soma size is not linked to input resistance for interneurons, contrary to the biophysical predictions of the 'size principle' for motor neurons. Next, we show that time constants are faster, excitatory currents stronger, and mixed potentials larger in lower resistance neurons, linking mixed synapse density to resting excitability. Using a computational model, we verify the impact of weighted electrical synapses on membrane properties, synaptic integration and the low-pass filtering and distribution of coupling potentials. We conclude differences in mixed synapse density can contribute to excitability underestimations and connectivity overestimations. The contribution of mixed synaptic inputs to resting excitability helps explain 'violations' of the size principle, where neuron size, resistance and recruitment order are unrelated.

Expression of myelin transcription factor 1 and lamin B receptor mediate neural progenitor fate transition in the zebrafish spinal cord pMN domain

The pMN domain is a restricted domain in the ventral spinal cord, defined by the expression of the olig2 gene. Though it is known that the pMN progenitor cells can sequentially generate motor neurons and oligodendrocytes, the lineages of these progenitors are controversial and how their progeny are generated is not well understood. Using single-cell RNA sequencing, here, we identified a previously unknown heterogeneity among pMN progenitors with distinct fates and molecular signatures in zebrafish. Notably, we characterized two distinct motor neuron lineages using bioinformatic analysis. We then went on to investigate specific molecular programs that regulate neural progenitor fate transition. We validated experimentally that expression of the transcription factor myt1 (myelin transcription factor 1) and inner nuclear membrane integral proteins lbr (lamin B receptor) were critical for the development of motor neurons and neural progenitor maintenance, respectively. We anticipate that the transcriptome features and molecular programs identified in zebrafish pMN progenitors will not only provide an in-depth understanding of previous findings regarding the lineage analysis of oligodendrocyte progenitor cells and motor neurons but will also help in further understanding of the molecular programming involved in neural progenitor fate transition.