Zebrafish Larvae Research Articles

Multiscale characterization of myelin distribution with polarized THG microscopy

Myelin is essential for axonal conduction and metabolic support. To better understand its role in health and disease, it is necessary to establish accurate methods for in situ mapping of myelin at scales ranging from submicrometer to centimeters. Third-harmonic generation (THG) microscopy has recently been proposed as an efficient label-free method to visualize myelin in thick and living tissue. However, the contrast mechanism of THG from myelinated axons is complex and poorly described, which has limited the development of THG as a quantitative probe of myelin distribution. Here, we present a systematic characterization and modeling of polarization-resolved THG (pTHG) signals from individual axons as a function of their diameter and myelin thickness, and we show that pTHG can be used to derive myelin scores in several biological systems. First, we confirm the sensitivity and specificity of the THG contrast for myelinated axons in mouse brain tissue and its ability to detect isolated micrometer-sized axons oriented both in-plane and out-of-plane. We then present a detailed characterization of the pTHG contrast of small and large axons in live zebrafish larvae at different developmental stages, and we demonstrate that pTHG detects early axon development in vivo. We show that classical models of coherent multiphoton microscopy fail to reproduce pTHG profiles of axons because they neglect myelin-induced optical aberrations, and we establish a numerical strategy based on finite-difference time-domain calculations that can accurately relate pTHG signal profiles to axonal diameter and myelin thickness. Finally, we illustrate the relevance of pTHG microscopy for characterizing myelin distribution at different scales in fixed mouse and human brain tissue.

Disruption of grin2A, an epilepsy-associated gene, produces altered spontaneous swim behavior in zebrafish

N-methyl-D-aspartate receptors (NMDARs) control synaptic plasticity and brain development in a manner determined by receptor subunit composition. Pathogenic variants in GRIN2A gene, encoding the NMDAR GluN2A subunit, can cause gain or loss of function of receptors containing the affected subunit, and are associated with intellectual disability and epilepsy in patients. While in-vitro studies of recombinant receptors have yielded some insights, animal experimental models are essential to better understand the relationship between the molecular pathology of the variants and the disease. Here we introduce a zebrafish model of GluN2A loss of function to study system-level effects of zebrafish grin2Aa and grin2Ab gene deletion. Our electrophysiological analysis revealed functional differences between receptors containing zebrafish GluN2Aa/b and GluN2Bb paralogs comparable to mammalian receptors containing GluN2A vs. GluN2B subunits. Both grin2Aa−/− and grin2Ab−/− , as well as double-knockout grin2A−/− zebrafish larvae showed increased locomotor activity in a novel environment. Proteomic analysis suggested that the relative proportion of GluN2B-containing NMDARs may be increased in grin2A mutant fish. Our results highlight fundamental similarities between zebrafish and mammalian NMDAR signaling and validate the use of zebrafish as a model organism to study the neurodevelopmental role of NMDARs. The newly created transgenic zebrafish strains complement the rodent models of GluN2A loss of function and can be used for high-throughput testing of pharmacological or genetic treatment strategies for patients with GRIN2A gene variants. Significance Statement N-methyl-D-aspartate receptors (NMDARs), critical for brain development and synaptic plasticity, are regulated by subunit composition. Pathogenic variants in the GRIN2A gene, encoding the GluN2A subunit, are linked to epilepsy and intellectual disability through altered receptor function. To explore system-level consequences of GluN2A loss, we developed zebrafish models lacking grin2Aa and grin2Ab genes. These mutants exhibited increased locomotor activity and possibly altered receptor subunit ratios, resembling features seen in mammalian GluN2A deficiency. Electrophysiological analysis confirmed functional distinctions between GluN2A- and GluN2B-containing zebrafish receptors, paralleling mammalian NMDAR properties. These results validate zebrafish as a model for GRIN2A-related disorders and establish a foundation for high-throughput screening of therapeutic strategies, complementing existing rodent models.

Toxicological mechanisms of gold nanoclusters in zebrafish embryos.

Toxicological mechanisms of gold nanoclusters in zebrafish embryos.

Structural and genetic determinants of zebrafish functional brain networks.

Network science has revealed universal brain connectivity principles across species. However, several macroscopic network features established in human neuroimaging studies remain underexplored at cellular scales in small animal models. Here, we use whole-brain calcium imaging in larval zebrafish to investigate the structural and genetic basis of functional brain networks. Mesoscopic functional connectivity (FC) robustly captures the individuality of larvae and reflects structural connectivity (SC) derived from single-neuron reconstructions. Several connectome properties, including diffusion mechanisms and indirect pathways, predict interregional correlations. SC and FC share a hierarchical modular architecture, with structural modules shaping spontaneous and stimulus-driven activity patterns. Visual stimuli and tail monitoring reveal a functional gradient that coincides with sensorimotor functions. Last, regional expression levels of specific genes predict interregional FC. Our findings reproduce key mammalian brain network features, demonstrating larval zebrafish as a powerful model for studying large-scale network phenomena in a small and optically accessible vertebrate brain.

Selenium Nanoplatform Engages Tranexamic Acid to Alleviate Ultraviolet B-Induced Skin Pigmentation via Inflammatory Response.

Skin is the largest organ of the body and serves as a barrier against various environmental threats, including ultraviolet (UV) radiation, which could lead to pigmentation and an increased risk of melanoma. Oxidative stress has been identified as a significant factor in UV-induced skin pigmentation, yet there still remain significant challenges in understanding its role in vivo. Selenium nanoparticles have exhibited multiple bioactivities in many fields, but their application in skin diseases is limited. In this work, the zebrafish screen was first carried out in a transgene zebrafish larva cocultured with Nano-Se, which demonstrated a whitening effect of Nano-Se on pigmentation caused by UVB exposure in vivo. Further study showed that Nano-Se could also boost the effectiveness of tranexamic acid, combined with the whitening effect in relieving UVB-induced skin pigmentation and damage in mice. In a word, the Nano-Se described in this study offers an efficient and practical approach for synthesizing bioactive selenium nanoplatforms, broadening the application of nanoselenium to combat UVB-induced skin damage, and potentially revitalizing research of nanoselenium in various human diseases.

Modeling high-risk pediatric cancers in zebrafish to inform precision therapy.

Despite advances in precision medicine, 30% of high-risk pediatric cancers lack an actionable molecular target, hindering effective treatment and impacting survival outcomes. While mouse patient-derived xenograft (PDX) models offer additional insights into clinical drug responses, delivering findings from these models within a clinically actionable timeframe remains challenging. This international collaboration between two national precision medicine programs demonstrates proof-of-principle that individualized larval zebrafish PDXs can robustly and rapidly assess clinical responses of high-risk child cancer patients. Retrospective zebrafish PDX testing was performed on tumor samples from ten pediatric patients with high-risk cancers. Drug responses in zebrafish models were correlated with clinical responses for each patient and directly compared with responses in cognate mouse PDX models. Responses to conventional and targeted therapies, administered as single agents or in combinations, were assessed. Zebrafish PDXs were successfully established from all ten patients and provided robust drug response data in every case, including from 3 patients whose tumor samples could not be engrafted in mice. Remarkably, zebrafish models accurately recapitulated patient responses for 11 of 12 treatment regimens. These findings highlight the potential of larval zebrafish PDX models to provide real-time, clinically relevant drug response data, supporting their potential use in prospective precision medicine studies.

Bilosomal Encapsulation of Binuclear Phosphino Ru(II)-Cu(II) Compounds Enhances Their Selectivity and Activity toward Lung and Prostate Cancers.

This study reports the synthesis, characterization, and biological evaluation of four novel heteronuclear Ru(II)-Cu(II) complexes with phosphine-fluoroquinolone conjugates. Structural analysis using X-ray diffraction, density functional theory calculations, and various spectroscopic techniques confirmed the stability and coordination geometries. The cytotoxicity was evaluated in vitro across multiple cancer cell lines (lung, breast, pancreatic, prostate) and noncancerous cells. In vivo toxicity was also assessed using a zebrafish (Danio rerio) larvae model. The results demonstrated that Ru(II)-Cu(II) complexes exhibit greater anticancer activity compared with cisplatin while almost being nontoxic toward control cells in vitro. Mechanistic studies revealed that their action involves nuclei accumulation, reactive oxygen species generation, lipid peroxidation, and mitochondrial membrane depolarization. The expression ratio of Bax to Bcl-2 mRNA correlates with apoptotic cell death. Additionally, encapsulation of the one Ru(II)-Cu(II) complex in bilosomes significantly improved its stability, selectivity, and therapeutic efficacy in three-dimensional (3D) cancer spheroid models.

Hinokitiol induces developmental and cardiovascular toxicity in zebrafish larvae and potential mechanisms.

Hinokitiol induces developmental and cardiovascular toxicity in zebrafish larvae and potential mechanisms.

Comparative immunomodulatory effects of chronic exposure to imidacloprid and thiamethoxam on the respiratory burst response in zebrafish and fathead minnow larvae.

Comparative immunomodulatory effects of chronic exposure to imidacloprid and thiamethoxam on the respiratory burst response in zebrafish and fathead minnow larvae.

Diverse prey capture strategies in teleost larvae.

Animal behavior is adapted to the sensory environment in which it evolved, while also being constrained by physical limits, evolutionary history, and developmental trajectories. The hunting behavior of larval zebrafish (Danio rerio), a cyprinid native to streams in Eastern India, has been well characterized. However, it is unknown if the complement and sequence of movements employed during prey capture by zebrafish is universal across freshwater teleosts. Here, we explore the syntax of prey capture behavior in larval fish belonging to the clade Percomorpha, whose last common ancestor with cyprinids lived ~240 mya. We compared the behavior of four cichlid species from Lake Tanganyika endemic to deep benthic parts of the lake (Lepidiolamprologus attenuatus, Lamprologus ocellatus, and Neolamprologus multifasciatus) or inhabiting rivers (Astatotilapia burtoni) with that of medaka (Oryzias latipes), a fish found in rice paddies in East Asia. Using high-speed videography and neural networks, we tracked eye movements and extracted swim kinematics during hunting from larvae of these five species. Notably, we found that the repertoire of hunting movements of cichlids is broader than that of zebrafish, but shares basic features, such as eye convergence, positioning of prey centrally in the binocular visual field, and discrete prey capture bouts, including two kinds of capture strikes. In contrast, medaka swim continuously, track the prey monocularly without eye convergence, and position prey laterally before capturing them with a side swing. This configuration of kinematic motifs suggests that medaka may judge distance to prey predominantly by motion parallax, while cichlids and zebrafish may mainly use binocular visual cues. Together, our study documents the diversification of locomotor and oculomotor adaptations among hunting teleost larvae.

Diverse prey capture strategies in teleost larvae

Animal behavior is adapted to the sensory environment in which it evolved, while also being constrained by physical limits, evolutionary history, and developmental trajectories. The hunting behavior of larval zebrafish (Danio rerio), a cyprinid native to streams in Eastern India, has been well characterized. However, it is unknown if the complement and sequence of movements employed during prey capture by zebrafish is universal across freshwater teleosts. Here, we explore the syntax of prey capture behavior in larval fish belonging to the clade Percomorpha, whose last common ancestor with cyprinids lived ~240 mya. We compared the behavior of four cichlid species from Lake Tanganyika endemic to deep benthic parts of the lake (Lepidiolamprologus attenuatus, Lamprologus ocellatus, and Neolamprologus multifasciatus) or inhabiting rivers (Astatotilapia burtoni) with that of medaka (Oryzias latipes), a fish found in rice paddies in East Asia. Using high-speed videography and neural networks, we tracked eye movements and extracted swim kinematics during hunting from larvae of these five species. Notably, we found that the repertoire of hunting movements of cichlids is broader than that of zebrafish, but shares basic features, such as eye convergence, positioning of prey centrally in the binocular visual field, and discrete prey capture bouts, including two kinds of capture strikes. In contrast, medaka swim continuously, track the prey monocularly without eye convergence, and position prey laterally before capturing them with a side swing. This configuration of kinematic motifs suggests that medaka may judge distance to prey predominantly by motion parallax, while cichlids and zebrafish may mainly use binocular visual cues. Together, our study documents the diversification of locomotor and oculomotor adaptations among hunting teleost larvae.

Unveiling the toxicological mechanisms and biological impacts of copper sulfide nanoparticles on zebrafish.

Nanoparticles (NPs) are increasingly released into the environment, posing risks to living organisms. While copper sulfide NPs have been extensively studied, the toxicity of copper sulfide (CuS) NPs remains underexplored. Owing to their widespread use, it is crucial to assess their biological effects. In this study, we used zebrafish embryos and larvae to evaluate CuS NP toxicity. Embryos were exposed to 20-100 µg/mL of CuS NPs under both dark and light conditions. Concentrations ≥60 µg/mL caused up to 85% mortality, along with developmental abnormalities such as bent tails and pericardial edema. Under light irradiation, CuS NPs significantly elevated reactive oxygen species (ROS) production. This was evidenced by upregulated expression of antioxidant genes sod1, sod2, cat, and gpx1 in exposed embryos. Our findings demonstrate a photoreactive toxicity mechanism: CuS NPs generate ROS under light, disrupting redox balance. This underscores the importance of environmental factors, like sunlight, in nanomaterial risk assessment. By revealing how CuS NPs affect aquatic organisms under realistic conditions, our study contributes to understanding the ecological risks these particles pose and highlights their potential implications for human health.

Metabolic Changes in Zebrafish Larvae Infected with Mycobacterium marinum: A Widely Targeted Metabolomic Analysis

Objectives: To explore the metabolic changes in zebrafish larvae after infection with Mycobacterium marinum, this study adopted a widely targeted metabolomic approach to analyze the changes in the overall metabolic profiles of zebrafish larvae infected for 5 days. Methods: Data were collected by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Mass spectrometry data were processed using Analyst 1.6.3 and MultiQuant 3.0.3 software, and multivariate statistical analysis was carried out. The KEGG database, HMDB database, and CHEBI database were used to screen and identify differential metabolites, and metabolic pathway enrichment analysis was performed through KEGG pathways. Results: A total of 329 metabolites were detected, among which 61 differential metabolites were screened. Specifically, 41 metabolites, such as kynurenine, isoallolithocholic acid, 2′-deoxyguanosine, indole-3-carboxaldehyde, and L-lactic acid, were downregulated, while 20 metabolites, such as L-palmitoylcarnitine, myristoyl-L-carnitine, dodecanoylcarnitine, 2-isopropyl-malic acid, and 2-methylsuccinic acid, were upregulated. KEGG metabolic pathway enrichment analysis indicated that these differential metabolites were mainly involved in metabolic pathways such as pyrimidine metabolism, nucleotide metabolism, the pentose phosphate pathway, and purine metabolism. Conclusions: This study demonstrated that significant changes occurred in multiple metabolites and metabolic pathways in zebrafish larvae after infection with M. marinum. The research results have improved the understanding of zebrafish as a model organism in the field of Mycobacterium research and laid a solid foundation for subsequent metabolomic-related research using zebrafish.

Modeling Pseudomonas aeruginosa-Staphylococcus aureus interactions in zebrafish to assess the host inflammatory response upon co-infection

Investigating the interplay between polymicrobial colonization and host response in the context of chronic infections is a complex issue. The interaction between Staphylococcus aureus and Pseudomonas aeruginosa, which frequently co-infect patients with chronic diseases like cystic fibrosis (CF), can be either competitive or coexistent. How these interaction states can influence the host response remains an open question for which relevant infection models are needed. In this study, we investigated co-infections by P. aeruginosa and S. aureus in zebrafish (Danio rerio), a non-mammalian model well established for the study of the host innate immune response. Using a wound infection method and a trio of strains co-isolated from a CF patient, we provide an in vivo co-infection model that recapitulates the competitive and coexistent bacterial interactions observed in vitro. The inflammatory response, monitored through the expression of specific cytokines at the infection site or in the whole larvae, was stronger with P. aeruginosa than S. aureus in the context of mono-infection. Upon co-infection, a competitive or coexistent interaction between P. aeruginosa and S. aureus appeared to slightly modulate this response. Moreover, the RT-qPCR profile of cytokine response observed in zebrafish larvae was similar to the one observed after infection of human lung epithelial cells. Thus, the zebrafish embryo appears as a relevant model to study persistent co-infection with P. aeruginosa and S. aureus, offering unique opportunities to address the host response in this polymicrobial context.

Multifaceted role of galanin in brain excitability.

Galanin is a neuropeptide, which is critically involved in homeostatic processes like controlling arousal, sleep, and regulation of stress. This extensive range of functions aligns with implications of galanin in diverse pathologies, including anxiety disorders, depression, and epilepsy. Here, we investigated the regulatory function of galanin on whole-brain activity in larval zebrafish using wide-field Ca2+ imaging. Combining this with genetic perturbations of galanin signaling and pharmacologically increasing neuronal activity, we are able to probe actions of galanin across the entire brain. Our findings demonstrate that under unperturbed conditions and during epileptic seizures, galanin exerts a sedative influence on the brain, primarily through the galanin receptor 1 a (galr1a). However, exposure to acute stressors like pentylenetetrazole (PTZ) compromises galanin's sedative effects, leading to overactivation of the brain and increased seizure occurrence. Interestingly, galanin's impact on seizures appears to be bidirectional, as it can both decrease seizure severity and increase seizure occurrence, potentially through different galanin receptor subtypes. This nuanced interplay between galanin and various physiological processes underscores its significance in modulating stress-related pathways and suggests its potential implications for neurological disorders such as epilepsy. Taken together, our data sheds light on a multifaceted role of galanin, where galanin regulates whole-brain activity but also shapes acute responses to stress.

Multimodal sensory overload in dopamine-deficient larval zebrafish leads to paradoxical kinesia.

Paradoxical kinesia-the temporary alleviation of motor deficits by powerful, urgent stimuli in Parkinson's disease (PD)-remains poorly understood at the neural circuit level. Through chemo-genetic ablation of tyrosine hydroxylase-expressing neurons in larval zebrafish and brain-wide calcium imaging under head-fixed, tail-free conditions, we uncovered a neural mechanism underlying this phenomenon. While catecholamine (CA)-deficient larvae exhibited severe locomotor deficits during free swimming, they showed paradoxical recovery of tail movements during whole-brain neural activity imaging. This locomotor recovery was accompanied by a significantly increased number of active neurons in the midbrain and hindbrain, but with reduced firing rates. Further analyses across 2158 anatomically defined regions allowed us to uncover a subset of regions, genes, and neurotransmitter types. GABAergic neurons were found to primarily account for the hyperactivity in the hindbrain, while glutamatergic neurons accounted for the hyperactivity in the midbrain. Hierarchical clustering of neuronal activity with tail movements revealed distinct motor- and non-motor-associated hyperactive clusters in the hindbrain and midbrain, respectively. We identified the Mesencephalic Locomotor Region (MLR) sandwiched between these domains, with enhanced glutamatergic firing rate and cholinergic activation. Furthermore, we found that Telencephalic corticotropin-releasing factor b (crhb) expressing neurons play a crucial role in mediating stress-response to the tectum, which in turn triggers a cascade of neuronal hyperactivity downstream via MLR. These findings reveal a neural mechanism that links stress-induced sensory processing with motor control systems in the absence of regulatory feedback from catecholaminergic neurons, suggesting a direct, unmodulated pathway that bypasses typical inhibitory controls.

Multifaceted role of galanin in brain excitability

Galanin is a neuropeptide, which is critically involved in homeostatic processes like controlling arousal, sleep, and regulation of stress. This extensive range of functions aligns with implications of galanin in diverse pathologies, including anxiety disorders, depression, and epilepsy. Here, we investigated the regulatory function of galanin on whole-brain activity in larval zebrafish using wide-field Ca2+ imaging. Combining this with genetic perturbations of galanin signaling and pharmacologically increasing neuronal activity, we are able to probe actions of galanin across the entire brain. Our findings demonstrate that under unperturbed conditions and during epileptic seizures, galanin exerts a sedative influence on the brain, primarily through the galanin receptor 1 a (galr1a). However, exposure to acute stressors like pentylenetetrazole (PTZ) compromises galanin’s sedative effects, leading to overactivation of the brain and increased seizure occurrence. Interestingly, galanin’s impact on seizures appears to be bidirectional, as it can both decrease seizure severity and increase seizure occurrence, potentially through different galanin receptor subtypes. This nuanced interplay between galanin and various physiological processes underscores its significance in modulating stress-related pathways and suggests its potential implications for neurological disorders such as epilepsy. Taken together, our data sheds light on a multifaceted role of galanin, where galanin regulates whole-brain activity but also shapes acute responses to stress.

Multifaceted role of galanin in brain excitability

Galanin is a neuropeptide, which is critically involved in homeostatic processes like controlling arousal, sleep, and regulation of stress. This extensive range of functions aligns with implications of galanin in diverse pathologies, including anxiety disorders, depression, and epilepsy. Here, we investigated the regulatory function of galanin on whole-brain activity in larval zebrafish using wide-field Ca2+ imaging. Combining this with genetic perturbations of galanin signaling and pharmacologically increasing neuronal activity, we are able to probe actions of galanin across the entire brain. Our findings demonstrate that under unperturbed conditions and during epileptic seizures, galanin exerts a sedative influence on the brain, primarily through the galanin receptor 1 a (galr1a). However, exposure to acute stressors like pentylenetetrazole (PTZ) compromises galanin’s sedative effects, leading to overactivation of the brain and increased seizure occurrence. Interestingly, galanin’s impact on seizures appears to be bidirectional, as it can both decrease seizure severity and increase seizure occurrence, potentially through different galanin receptor subtypes. This nuanced interplay between galanin and various physiological processes underscores its significance in modulating stress-related pathways and suggests its potential implications for neurological disorders such as epilepsy. Taken together, our data sheds light on a multifaceted role of galanin, where galanin regulates whole-brain activity but also shapes acute responses to stress.

Spatial-offset pump-probe imaging of nonradiative dynamics at optical resolution.

Nonradiative photothermal (PT) and photoacoustic (PA) processes have found widespread applications in imaging, stimulation, and therapy. Mapping the generation and propagation of PA and PT waves with high resolution is important to elucidate the interaction between nonradiative fields and biological systems. To this end, we introduce spatial-offset pump-probe imaging (SOPPI). By spatially offsetting the pump beam and the probe beam, SOPPI allows simultaneous imaging of PA/PT wave propagation with nanosecond temporal resolution, micrometer spatial resolution, 65-megahertz detection bandwidth, and a sensitivity of 9.9-pascal noise equivalent pressure. First, the PA and PT evolution from a fiber emitter and the subsequent wave interaction with a mouse skull and brain slices are demonstrated. Then, using water as the absorber, a wavelength-dependent PA/PT generation, evanescent wave-generated PA, and a back-propagated acoustic Mach cone with a tapered fiber are recorded. At last, a SOPPI-PACT is developed to reconstruct the pigment distribution inside a zebrafish larva with high precision and signal-to-noise ratio.

Real-Time Monitoring of Mitochondrial pH in HeLa Cells, Drosophila melanogaster, and Zebrafish Larvae Using BODIPY-Based Ratiometric Fluorescent Probes.

Three BODIPY-based fluorescent probes, AH+, BH+, and CH+, were synthesized for ratiometric pH sensing in living cells, fruit flies, and zebrafish larvae. These probes were designed to target mitochondrial environments by functionalizing the BODIPY core with various substituent groups that tune the pH sensitivity. The probes exhibited strong ratiometric fluorescence changes with pKa values (AH+, 7.3; BH+, 7.5; CH+, 7.2) suitable for mitochondrial pH detection. Theoretical calculations supported these findings by establishing the geometries and electronic transitions and also resulted in the derivation of their pKa values. Confocal imaging confirmed mitochondrial accumulation of these probes in HeLa cells, facilitating broad-range pH monitoring across a wide pH spectrum (3.5 to 9.1). These ratiometric pH sensors display good reversibility and response times under varying pH conditions. In application, the probe AH+ was employed to monitor pH fluctuations under conditions of oxidative stress and nutrient deprivation. Dual-channel cell imaging revealed a pH-dependent fluorescence shift with precise transitions, demonstrating the feasibility of real-time monitoring of the mitochondrial membrane potential in living cells. Furthermore, the probe AH+ effectively visualized pH changes in Drosophila melanogaster and zebrafish larvae, further supporting its applicability across diverse biological systems. We demonstrate that a fluorescence ratiometric intensity graph for probe AH+ can be effectively employed to determine pH values within the mitochondria of HeLa cells.