Methods: A bioinformatic protocol for rapid analysis of zebrafish embryo photo-motory responses (PMR) in neurotoxicity testing.

Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals. Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos.

An Open-Source Programmable Interface for Sensory-Motor Biotests with Zebrafish (Danio rerio).

Assessment of animals' sensory-motor functions requires precise and electronically controlled stimuli to induce and quantify specific behavioral phenotypes. However, accessible and inexpensive tools for conducting diverse sensory-motor biotests with fish are lacking. In this work, we present an open-source software and hardware interface that enables automated delivery of three independent and fully programmable stimuli for behavioral bioassays. We demonstrate the proof-of-concept application of this low-cost technology in establishing reproducible fear responses using a mechanical tap-startle stimulus in larval zebrafish. This response is characterized by a sudden burst of motion in response to a nondirectional mechanical stimulus delivered to the fish chamber. We propose that the simplicity and flexibility of this interface offer innovative opportunities for studying sensory-motor functions in various fields, including neurobiology, neuropharmacology, neurotoxicology, and aquatic ecotoxicology.

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.

Toward Real-Time Animal Tracking with Integrated Stimulus Control for Automated Conditioning in Aquatic Eco-Neurotoxicology.

Aquatic eco-neurotoxicology is an emerging field that requires new analytical systems to study the effects of pollutants on animal behaviors. This is especially true if we are to gain insights into one of the least studied aspects: the potential perturbations that neurotoxicants can have on cognitive behaviors. The paucity of experimental data is partly caused by a lack of low-cost technologies for the analysis of higher-level neurological functions (e.g., associative learning) in small aquatic organisms. Here, we present a proof-of-concept prototype that utilizes a new real-time animal tracking software for on-the-fly video analysis and closed-loop, external hardware communications to deliver stimuli based on specific behaviors in aquatic organisms, spanning three animal phyla: chordates (fish, frog), platyhelminthes (flatworm), and arthropods (crustacean). The system's open-source software features an intuitive graphical user interface and advanced adaptive threshold-based image segmentation for precise animal detection. We demonstrate the precision of animal tracking across multiple aquatic species with varying modes of locomotion. The presented technology interfaces easily with low-cost and open-source hardware such as the Arduino microcontroller family for closed-loop stimuli control. The new system has potential future applications in eco-neurotoxicology, where it could enable new opportunities for cognitive research in diverse small aquatic model organisms.

A miniaturized electrothermal array for rapid analysis of temperature preference behaviors in ecology and ecotoxicology.

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.

Targeted Isolation of Antibiotic Brominated Alkaloids from the Marine Sponge Pseudoceratina durissima Using Virtual Screening and Molecular Networking.

Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8).

Sensory-Motor Perturbations in Larval Zebrafish (Danio rerio) Induced by Exposure to Low Levels of Neuroactive Micropollutants during Development.

Due to increasing numbers of anthropogenic chemicals with unknown neurotoxic properties, there is an increasing need for a paradigm shift toward rapid and higher throughput behavioral bioassays. In this work, we demonstrate application of a purpose-built high throughput multidimensional behavioral test battery on larval stages of Danio rerio (zebrafish) at 5 days post fertilization (dpf). The automated battery comprised of the established spontaneous swimming (SS), simulated predator response (SPR), larval photomotor response (LPR) assays as well as a new thermotaxis (TX) assay. We applied the novel system to characterize environmentally relevant concentrations of emerging pharmaceutical micropollutants including anticonvulsants (gabapentin: 400 ng/L; carbamazepine: 3000 ng/L), inflammatory drugs (ibuprofen: 9800 ng/L), and antidepressants (fluoxetine: 300 ng/L; venlafaxine: 2200 ng/L). The successful integration of the thermal preference assay into a multidimensional behavioral test battery provided means to reveal ibuprofen-induced perturbations of thermal preference behaviors upon exposure during embryogenesis. Moreover, we discovered that photomotor responses in larval stages of fish are also altered by the as yet understudied anticonvulsant gabapentin. Collectively our results demonstrate the utility of high-throughput multidimensional behavioral ecotoxicity test batteries in prioritizing emerging risks associated with neuroactive drugs that can perturb neurodevelopment. Moreover, we showcase the added value of thermotaxis bioassays for preliminary screening of emerging contaminants.

Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

Accelerating Chemobehavioral Phenotypic Screening in Neurotoxicology Using a Living Embryo Array System.

Large-scale chemobehavioral phenotyping with zebrafish embryos is a promising avenue for accelerated neurotoxicity testing and discovery of behavior-modifying neuroceuticals. These strategies are hampered by lack of effective embryo in-test positioning, wide-field imaging, and high-throughput bioinformatic analytics. In this study, we demonstrate advantages of using custom large-density embryo arrays in conjunction with an open-source ultra-high-definition video imaging system. Moreover, we present a high-throughput bioinformatics workflow for rapid behavioral analysis of large cohorts of specimens in photomotor response bioassays. The system validation was showcased in a proof-of-concept neurotoxicity analysis.

Novel preclinical model for CDKL5 deficiency disorder.

Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies. This article has an associated First Person interview with the first author of the paper.

Inhibition of Amyloid Aggregation and Toxicity with Janus Iron Oxide Nanoparticles.

Amyloid aggregation is a ubiquitous form of protein misfolding underlying the pathologies of Alzheimer's disease (AD), Parkinson's disease (PD) and type 2 diabetes (T2D), three primary forms of human amyloid diseases. While much has been learned about the origin, diagnosis and management of these neurological and metabolic disorders, no cure is currently available due in part to the dynamic and heterogeneous nature of the toxic oligomers induced by amyloid aggregation. Here we synthesized beta casein-coated iron oxide nanoparticles (ßCas IONPs) via a BPA-P(OEGA-b-DBM) block copolymer linker. Using a thioflavin T kinetic assay, transmission electron microscopy, Fourier transform infrared spectroscopy, discrete molecular dynamics simulations and cell viability assays, we examined the Janus characteristics and the inhibition potential of ßCas IONPs against the aggregation of amyloid beta (Aß), alpha synuclein (αS) and human islet amyloid polypeptide (IAPP) which are implicated in the pathologies of AD, PD and T2D. Incubation of zebrafish embryos with the amyloid proteins largely inhibited hatching and elicited reactive oxygen species, which were effectively rescued by the inhibitor. Furthermore, Aß-induced damage to mouse brain was mitigated in vivo with the inhibitor. This study revealed the potential of Janus nanoparticles as a new nanomedicine against a diverse range of amyloid diseases.

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

RNA-induced inflammation and migration of precursor neurons initiates neuronal circuit regeneration in zebrafish.

Tissue regeneration and functional restoration after injury are considered as stem- and progenitor-cell-driven processes. In the central nervous system, stem cell-driven repair is slow and problematic because function needs to be restored rapidly for vital tasks. In highly regenerative vertebrates, such as zebrafish, functional recovery is rapid, suggesting a capability for fast cell production and functional integration. Surprisingly, we found that migration of dormant "precursor neurons" to the injury site pioneers functional circuit regeneration after spinal cord injury and controls the subsequent stem-cell-driven repair response. Thus, the precursor neurons make do before the stem cells make new. Furthermore, RNA released from the dying or damaged cells at the site of injury acts as a signal to attract precursor neurons for repair. Taken together, our data demonstrate an unanticipated role of neuronal migration and RNA as drivers of neural repair.

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA.

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgshΔex5-6 zebrafish is largely dependent on interleukin-1ß and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

Cellular and Molecular Characterization of the Effects of the Zebrafish Embryo Genotyper Protocol.

The Zebrafish Embryo Genotyper (ZEG) device provides a promising tool for genotyping live embryos. Although the gross morphology and survival of embryos after the use of ZEG are unaffected, the cellular and molecular effects of the ZEG protocol remain unknown. To address this, we have examined the integrity of specific tissues, and evaluated the expression of stress-responsive genes to determine the impact of the ZEG protocol. Our analyses reveal that although ZEG results in a low-level acute stress response, no long-lasting effects are evident, supporting its utilization for a variety of downstream assays.

TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages.

Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.

Neurogenesis in the inner ear: the zebrafish statoacoustic ganglion provides new neurons from a Neurod/Nestin-positive progenitor pool well into adulthood.

The vertebrate inner ear employs sensory hair cells and neurons to mediate hearing and balance. In mammals, damaged hair cells and neurons are not regenerated. In contrast, hair cells in the inner ear of zebrafish are produced throughout life and regenerate after trauma. However, it is unknown whether new sensory neurons are also formed in the adult zebrafish statoacoustic ganglion (SAG), the sensory ganglion connecting the inner ear to the brain. Using transgenic lines and marker analysis, we identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In particular, we analyze a Neurod/Nestin-positive progenitor pool that produces large amounts of new neurons at juvenile stages, which transitions to a quiescent state in the adult SAG. Moreover, BrdU pulse chase experiments reveal the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestin-positive progenitor pool at adult stages. Taken together, our study represents the first comprehensive characterization of the adult zebrafish SAG showing that zebrafish, in sharp contrast to mammals, display continued neurogenesis in the SAG well beyond embryonic and larval stages.

Bioinspired polynorepinephrine nanoparticles as an efficient vehicle for enhanced drug delivery.

An innovative drug delivery vehicle based on polynorepinephrine (PNE) with controllable size modification, high delivery efficacy and low cytotoxicity is presented. Highly monodisperse PNE nanoparticles are fabricated by the autoxidation of norepinephrine monomers in an alkaline water/ethanol mixture via stirring at room temperature. We demonstrated the facile optimization of particle size to enhance particle stability and biocompatibility by varying solvent and monomer dosage. To demonstrate the suitability and potential application of PNE particles in cancer therapy, we show that these particles are biocompatible in vitro with HeLa cells and in vivo in zebrafish embryos. After loading the anti-cancer chemotherapy drug doxorubicin (DOX) into the PNE nanoparticles, a consistent and pH responsive drug release profile of DOX was achieved in different environmental conditions. It was found that DOX loaded PNE nanoparticles (PNE/DOX) exhibit much higher pharmaceutical cytotoxicity than free DOX on HeLa cells. Furthermore, the amount of drug released was significantly enhanced in acidic environments that mimic the pH of extracellular tumour microenvironments. Taken together, the PNE nanoparticles represent a new class of melanin particles with promising potential in drug delivery and as a therapeutic platform for cancer treatment.

Accelerated brain aging towards transcriptional inversion in a zebrafish model of the K115fs mutation of human PSEN2.

BACKGROUND: The molecular changes involved in Alzheimer's disease (AD) progression remain unclear since we cannot easily access antemortem human brains. Some non-mammalian vertebrates such as the zebrafish preserve AD-relevant transcript isoforms of the PRESENILIN genes lost from mice and rats. One example is PS2V, the alternative transcript isoform of the PSEN2 gene. PS2V is induced by hypoxia/oxidative stress and shows increased expression in late onset, sporadic AD brains. A unique, early onset familial AD mutation of PSEN2, K115fs, mimics the PS2V coding sequence suggesting that forced, early expression of PS2V-like isoforms may contribute to AD pathogenesis. Here we use zebrafish to model the K115fs mutation to investigate the effects of forced PS2V-like expression on the transcriptomes of young adult and aged adult brains. METHODS: We edited the zebrafish genome to model the K115fs mutation. To explore its effects at the molecular level, we analysed the brain transcriptome and proteome of young (6-month-old) and aged (24-month-old) wild type and heterozygous mutant female sibling zebrafish. Finally, we used gene co-expression network analysis (WGCNA) to compare molecular changes in the brains of these fish to human AD. RESULTS: Young heterozygous mutant fish show transcriptional changes suggesting accelerated brain aging and increased glucocorticoid signalling. These early changes precede a transcriptional 'inversion' that leads to glucocorticoid resistance and other likely pathological changes in aged heterozygous mutant fish. Notably, microglia-associated immune responses regulated by the ETS transcription factor family are altered in both our zebrafish mutant model and in human AD. The molecular changes we observe in aged heterozygous mutant fish occur without obvious histopathology and possibly in the absence of Aß. CONCLUSIONS: Our results suggest that forced expression of a PS2V-like isoform contributes to immune and stress responses favouring AD pathogenesis. This highlights the value of our zebrafish genetic model for exploring molecular mechanisms involved in AD pathogenesis.