Zebrafish Larvae Research Articles

Assessment of the Novel Anti-Seizure Potential of Validamycin A Using Zebrafish Epilepsy Model.

Epilepsy is a prevalent neurological disorder characterized by recurrent seizures. Validamycin A (VA) is an antibiotic fungicide that inhibits trehalase activity and is widely used for crop protection in agriculture. In this study, we identified a novel function of VA as a potential anti-seizure medication in a zebrafish epilepsy model. Electroencephalogram (EEG) analysis demonstrated that VA reduced pentylenetetrazol (PTZ)-induced seizures in the brains of larval and adult zebrafish. Moreover, VA reduced PTZ-induced irregular movement in a behavioral assessment of adult zebrafish. The developmental toxicity test showed no observable anatomical alteration when the zebrafish larvae were treated with VA up to 10 µM within the effective range. The median lethal dose of VA in adult zebrafish was > 14,000 mg/kg. These results imply that VA does not demonstrate observable toxicity in zebrafish at concentrations effective for generating anti-seizure activity in the EEG and alleviating abnormal behavior in the PTZ-induced epileptic model. Furthermore, the effectiveness of VA was comparable to that of valproic acid. These results indicate that VA may have a potentially safer anti-seizure profile than valproic acid, thus offering promising prospects for its application in agriculture and medicine.

Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From β-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic β-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 β-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost β-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for β-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering β-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing β-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of β-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting β-cell functionality.

Myclobutanil induces cardiotoxicity in developing zebrafish larvae by initiating oxidative stress and apoptosis: The protective role of curcumin

Myclobutanil (MYC) is a common triazole fungicide widely applied in agriculture. MYC extensively exists in the natural environment and can be detected in organisms. However, little is known about MYC-induced embryonic developmental damage. This study aimed to unravel the cardiotoxicity of MYC and the underlying mechanisms, as well as the cardioprotective effect of curcumin (CUR, an antioxidant polyphenol) using the zebrafish model. Here, zebrafish embryos were exposed to MYC at concentrations of 0, 0.5, 1 and 2 mg/L from 4 to 96 h post fertilization (hpf) and cardiac development was assessed. As results, MYC reduced the survival and hatching rate, body length and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal cardiac morphology and function in myl7:egfp transgenic zebrafish, and downregulated cardiac developmental genes. MYC promoted oxidative stress through excessive reactive oxygen species (ROS) accumulation and suppressed the activities of antioxidant enzymes, triggering cardiomyocytic apoptosis via upregulated expression of apoptosis-related genes. These adverse toxicities could be significantly ameliorated by the antioxidant properties of CUR, indicating that CUR rescued MYC-induced cardiotoxicity by inhibiting oxidative stress and apoptosis. Overall, our study revealed the potential mechanisms of oxidative stress and apoptosis in MYC-induced cardiotoxicity in zebrafish and identified the cardioprotection of CUR in this pathological process.

The RGK protein rrad regulates cardiac calcium levels in zebrafish

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain Background/Introduction High voltage-activated Ca2+ (CaV) channels are essential for excitation-contraction coupling. RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind CaV β-subunits and profoundly inhibit CaV1/CaV2 channels. A recent study showed that Rad inhibits CaV1.2 channels in cardiomyocytes, thus mediating the enhanced inotropy and chronotropy by β-adrenergic agonists during the fight-or-flight response. The zebrafish has emerged as a useful in vivo animal model in cardiovascular physiology and pathophysiology. The role of zebrafish rrad, the orthologe of the human RAD, in the regulation of cardiac Ca2+ homeostasis in zebrafish has not been investigated. Purpose The aim of this study was to investigate the role of rrad in cardiac Ca2+ regulation in zebrafish. Methods We evaluated the cardiac Ca2+ dynamics, heart rhythm, and hemodynamics in transgenic zebrafish larvae that overexpress rrad in the heart, as well as in heterozygous rrad knockout larvae. Measurements were done in the beating heart of 3 dpf transgenic zebrafish larvae expressing the fluorescent Ca2+ biosensor Twitch-4. Results In some larvae, overexpression of rrad induced a severe enlargement of the atrium with pericardial edema and a high mortality rate. In addition, the ventricle displayed low Ca2+ levels and reduced contractility. The remaining larvae had no altered morphology, but the ventricular Ca2+ levels were reduced. On the other hand, preliminary results showed that down regulation of rrad in zebrafish larvae increased atrial and ventricular Ca2+ levels, like what was found in Rad knockout mice. Conclusion Taken together, these findings suggest that rrad in the zebrafish heart, as in mammals, is an inhibitor of the Ca2+ entry, presumably through CaV1/CaV2 channels, and demonstrate the zebrafish's utility as a model to study the involvement of rrad in cardiac function.

In vivo calcium and voltage mapping of the zebrafish heart

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation Flanders (FWO) Purpose Inherited cardiac arrhythmias are characterized by a disturbance in the normal electrical functioning of the heart due to a dysfunction of cardiac ion channels, accessory proteins and/or structural components. This complex process is difficult to model in vitro, while in vivo mouse models differ substantially from humans with regards to cardiac electrophysiology. Thus, there is a need for alternative disease models in cardiac arrhythmia research. The zebrafish heart shows remarkable similarity to humans in its electrocardiogram and action potential (AP) morphology. Due to the optical translucency of zebrafish larvae, it is possible visualize the cardiac calcium and voltage dynamics across their entire hearts in vivo by use of genetically encoded calcium and voltage indicators (GECI and GEVI). We combined the fast kinetics of a next generation voltage reporter (Ace2N-mNeon) with a spectrally compatible calcium sensor (R-GECO) to create a sensitive high throughput method for in vivo characterization of the cardiac AP. Methods Ace2N-mNeon and R-GECO were expressed under control of a myocardial specific promoter via the Tol2kit system. The effect of sensor expression on normal physiology was assessed by patch clamp experiments. Fluorescence recordings were obtained with a Leica SP8 DLS microscope and a custom-built light sheet microscope. Our zebrafish sensor line was validated by experiments with quinidine drug exposure, as well as a novel model for long QT syndrome (LQTS) with a knock-in (KI) mutation in the cacna1c gene. Results Sensor expression did not significantly alter cardiac electrophysiology. The tg(myl7:Ace2N-mNeon/R-GECO) line demonstrated alterations in the heart rate, depolarization and repolarization induced by quinidine exposure. We observed a significantly prolonged ventricular repolarization in the LQTS KI line. Three-dimensional optical maps of AP characteristics across the entire zebrafish heart exhibited the physiological characteristics of the zebrafish cardiac electrophysiology, with activation initiated at the atrial venous pole and propagated towards the ventricular outflow tract, prolonged repolarization in the ventricles compared to the atria and slowed depolarization in the atrio-ventricular canal. Conclusion By expressing Ace2N-mNeon and R-GECO in the zebrafish heart, we were able to accurately detect drug- and mutation induced alterations in the cardiac AP, as well as AP characteristics across the entire heart, without compromising normal cardiac electrophysiology. Our transgenic line will provide a promising assay for future studies of cardiac arrhythmia.

Loss of pitx2c causes early alterations in atrial calcium handling in zebrafish

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain (PID2019-111456RB-100); Consejería de Educación, Cultura y Deportes, Junta de Comunidades de Castilla-La Mancha (SBPLY/19/180501/000223, co-funded by EU FEDER-ERDF) and grants for research groups from University of Castilla-La Mancha (2019-GRIN-27019, 2020-GRIN-29186 and 2022-GRIN-34301, co-funded by EU FEDER-ERDF) Introduction Atrial fibrillation (AF) is the most common cardiac arrhythmia in humans. Genome-wide association studies revealed that AF is related to sequence variants in chromosomic regions in close proximity to the transcription factor PITX2, involved in cardiac development and asymmetry, and in morphogenesis of pacemaker regions such as the sinoatrial region and the atrioventricular canal, which receive significant input from the autonomic nervous system. AF patients showed reduced PITX2 mRNA levels. PITX2-/- atrial tissue showed lower expression of CACNA1C accompanied by lower L-type Ca2+ current density. A recent study revealed AF-like phenotypes, including fibrosis, structural and metabolic alterations, arrhythmia, and cardiac conduction defects in adult pitx2c -/- zebrafish. Nevertheless, Ca2+ handling alterations remain unexplored in this zebrafish model. Purpose In this study we investigated cardiac Ca2+ handling in pitx2c deficient zebrafish. Methods We used the transgenic zebrafish line Tg(myl7:Twitch-4) expressing a fluorescent Ca2+ indicator in the heart. We outcrossed pitx2c+/- fish with Tg(myl7:Twitch-4) pitx2c+/- and studied Ca2+ dynamics and cardiac function by videomicroscopy in the beating hearts of 5 days post fertilization (dpf) larvae. Larvae were treated with 100 µM carbachol to assess differential response to parasympathetic stimulation in pitx2c deficient larvae. Results We observed sinoatrial pauses in 40 % of pitx2c+/- larvae accompanied by an increase in heart rate variability. In addition, pitx2c+/- and pitx2c-/- larvae at 5 dpf showed differential response to carbachol, a cholinergic agonist, compared to their wild-type siblings. Moreover, carbachol significantly decreased heart rate in pitx2c+/- and pitx2c-/- 5dpf larvae compared to siblings. Furthermore, we also observed a reduction of the amplitude of Ca2+ transients due to a decrease in the systolic Ca2+ levels in the atrium of pitx2c+/- larvae. The rise time of the atrial Ca2+ transient was prolonged, with a decrease in the rise slope. These data suggest a lower Ca2+ entry in the atrium. Interestingly, pitx2c+/- and wild-type larvae showed similar ventricular Ca2+ handling and contractility. Conclusion These results suggest that loss of pitx2c induce early Ca2+ alterations in the atrium as well as sinoatrial dysfunction in zebrafish during embryogenesis. Furthermore, pitx2c deficiency in zebrafish larvae possibly alters autonomic innervation leading to differentiated cardiac response to carbachol.

MTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions

BackgroundThe larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated.Materials and resultsThis study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration.ConclusionIn summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

Nkx1.2 deletion decreases fat production in zebrafish.

This study aimed to investigate the role of Nkx1-2, a transcription factor with the NK homeobox domain, in the regulation of fat production. Gene expression was analyzed using quantitative real-time polymerase chain reaction or transcriptome sequencing. CRISPR/Cas9 technology was employed to generate nkx1.2 knockout zebrafish and nkx1.2-deleted 3T3-L1 cells. Lipid droplet production in zebrafish larvae was visually quantified using Nile red staining, whereas lipid droplets in 3T3-L1 cells were stained with Oil red O. The binding of Nkx1-2 to the promoter was verified through an electrophoretic mobility shift assay experiment. Nkx1-2 plays crucial roles in the regulation of fat production in zebrafish. Knockout of nkx1.2 in zebrafish leads to weight loss, accompanied by significantly reduced lipid droplet production and decreased visceral and liver fat content. Furthermore, genes related to lipid biosynthesis are significantly downregulated. In 3T3-L1 preadipocytes, Nkx1-2 induces differentiation into mature adipocytes by binding to the cebpa promoter, thereby activating its transcription. Additionally, the expression of nkx1.2 is regulated by the p38 MAPK, JNK, or Smad2/3 signaling pathways in 3T3-L1 cells. Our findings suggest that Nkx1-2 functions as a positive regulator of fat production, playing a critical role in adipocyte differentiation and lipid biosynthesis.

Behavioral Effects of the Mixture and the Single Compounds Carbendazim, Fipronil, and Sulfentrazone on Zebrafish (Danio rerio) Larvae.

Pesticides are often detected in freshwater, but their impact on the aquatic environment is commonly studied based on single compounds, underestimating the potential additive effects of these mixtures. Even at low concentrations, pesticides can negatively affect organisms, altering important behaviors that can have repercussions at the population level. This study used a multi-behavioral approach to evaluate the effects of zebrafish larvae exposure to carbendazim (C), fipronil (F), and sulfentrazone (S), individually and mixed. Five behavioral tests, thigmotaxis, touch sensitivity, optomotor response, bouncing ball test, and larval exploratory behavior, were performed to assess potential effects on anxiety, fear, and spatial and social interaction. Significant changes were observed in the performance of larvae exposed to all compounds and their mixtures. Among the single pesticides, exposure to S produced the most behavioral alterations, followed by F and C, respectively. A synergistic effect between the compounds was observed in the C + F group, which showed more behavioral effects than the groups exposed to pesticides individually. The use of behavioral tests to evaluate pesticide mixtures is important to standardize methods and associate behavioral changes with ecologically relevant events, thus creating a more realistic scenario for investigating the potential environmental impacts of these compounds.

Isavuconazole Induces Neurodevelopment Defects and Motor Behaviour Impairment in Zebrafish Larvae.

Isavuconazole is a broad-spectrum antifungal drug used for the treatment of serious infections caused by invasive aspergillosis and mucormycosis in adults. With the continuous use of this drug, its safety and environmental impact have received increasing attention. However, information on the adverse effects of the drug is very limited. Fish is a particularly important model for assessing environmental risks. In this study, the aquatic vertebrate zebrafish was used as a model to study the toxic effects and mechanisms of isavuconazole. We exposed zebrafish embryos to 0.25, 0.5, and 1mg/L of isavuconazole 6h after fertilization. The results showed that at 72 hpf, isavuconazole exposure reduced heart rate, body length, and survival of zebrafish embryos compared to controls. Secondly, when isavuconazole reached a certain dose level (0.25mg/L), it caused morphological changes in the Tg(elavl3:eGFP) transgenic fish line, with the head shrunk, the body bent, the fluorescence intensity becoming weaker, the abnormal motor behaviour, etc. At the same time, exposure of zebrafish embryos to isavuconazole downregulated acetylcholinesterase (AchE) and adenosine triphosphate (ATPase) activities but upregulated oxidative stress, thereby disrupting neural development and gene expression of neurotransmitter pathways. In addition, astaxanthin partially rescued the neurodevelopmental defects of zebrafish embryos by downregulating oxidative stress. Thus, our study suggests that isavuconazole exposure may induce neurodevelopment defects and behavioural disturbances in larval zebrafish.

Non-negligible Toxicity to Fish in the Early Life Stages Triggered by Aqueous Leachate of Takeaway Plastic Containers.

Ordering takeout is a growing social phenomenon and may raise public health concerns. However, the associated health risk of compounds leaching from plastic packaging is unknown due to the lack of chemical and toxicity data. In this study, 20 chemical candidates were tentatively identified in the environmentally relevant leachate from plastic containers through the nontargeted chemical analysis. Three main components with high responses and/or predicted toxicity were further verified and quantified, namely, 3,5-di-tert-butyl-4-hydroxycinnamic acid (BHC), 2,4-di-tert-butylphenol (2,4-DTBP), and 9-octadecenamide (oleamide). The toxicity to zebrafish larvae of BHC, a degradation product of a widely used antioxidant Irganox 1010, was quite similar to that of the whole plastic leachate. In the same manner, RNA-seq-based ingenuity analysis showed that the affected canonical pathways of zebrafish larvae were quite comparable between BHC and the whole plastic leachate, i.e., highly relevant to neurological disease, metabolic disease, and even behavioral disorder. Longer-term exposure (35 days) did not cause any effect on adult zebrafish but led to decreased hatching rate and obvious neurotoxicity in zebrafish offspring. Collectively, this study strongly suggests that plastic containers can leach out a suite of compounds causing non-negligible impacts on the early stages of fish via direct or parental exposure.

A highly sensitive and selective fluorescent “on–off-on” peptide-based probe for sequential detection of Hg2+ and S2− ions: Applications in living cells and zebrafish imaging

Mercury ions (Hg2+) and sulfur ions (S2−), have caused serious harm to the ecological environment and human health as two kinds of highly toxic pollutants widely used. Therefore, the visual quantitative determination of Hg2+ and S2− is of great significance in the field of environmental monitoring and medical therapy. In this study, a novel fluorescent “on–off-on” peptide-based probe DNC was designed and synthesized using dipeptide (Asn-Cys-NH2) as the raw material via solid phase peptide synthesis (SPPS) technology with Fmoc chemistry. DNC displayed high selectivity in the recognition of Hg2+, and formed non-fluorescence complex (DNC-Hg2+) through 2:1 binding mode. Notably, DNC-Hg2+ complex generated in situ was used as relay response probe for highly selective sequential detection of S2− through reversible formation-separation. DNC achieved highly sensitive detection of Hg2+ and S2− with the detection limits (LODs) of 8.4 nM and 5.5 nM, respectively. Meanwhile, DNC demonstrated feasibility for Hg2+ and S2− detections in two water samples, and the considerable recovery rate was obtained. More importantly, DNC showed excellent water solubility and low toxicity, and was successfully used for consecutive discerning Hg2+ and S2− in test strips, living cells and zebrafish larvae. As an effective visual analysis method in the field, smartphone RGB Color Picker APP realized semi-quantitative detections of Hg2+ and S2− without the need for complicated device.

#2407 Multiomics analysis of isolated zebrafish glomeruli to unravel the impact of alternative splicing in glomerular diseases

Abstract Background and Aims Alternative splicing (AS) is a known key factor in gene regulation. There is evidence that changes in the alternative mRNA splicing can lead to the onset of various diseases. However, the potential role of AS in the pathogenesis of glomerular diseases has only been investigated to a very limited extent. In the past, the zebrafish larva (Danio rerio) has been established as an animal model for the in vivo investigation of kidney diseases, since the glomeruli of zebrafish larvae are genetically, molecularly, and functionally similar to those of mammals. As part of the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project, we wanted to identify specific AS events that might be involved in the development of focal segmental glomerulosclerosis (FSGS). Method For all experiments, the double transgenic strain [Tg (nphs2: GAL4-VP16); (UAS:Eco.nfsB-mCherry)] was used. At 4 dpf (days post fertilization), the zebrafish larvae were divided into two groups. The control group was treated with 0.1% dimethyl sulfoxide (DMSO) in E3 medium. To induce FSGS, zebrafish larvae were treated with 50 nM nifurpirinol (NFP) in 0.1% DMSO in E3 medium for 24 hours. Glomeruli were manually isolated at 5 and 6 dpf and analyzed together with lysates of whole larvae by LC-MS/MS and RNA-Seq. To identify AS events, RNA-Seq data were analyzed by MAJIQ, Multivariate Analysis of Transcript Splicing (rMATS), leafcutter, Whippet as well as IsoformSwitchAnalyzeR. Gene set enrichment analysis (GSEA) identified proteins and genes which were classified according to their related biological processes. Results Proteomics and transcriptomics showed significant differences in the glomerular protein and transcript levels between NFP-treated larvae and the control group. The abundance of the podocyte-specific protein nephrin as well as the podocin promotor-driven expression of the fluorescent dye mCherry was significantly reduced after NFP treatment confirming the onset of the injury. Subsequent analyses identified more than 1000 transcripts that were significantly differentially expressed, as well as several splicing events in the larvae with a FSGS-like damage. There was also a marked shift in isoform patterns in larvae with FSGS. Gene set enrichment analysis indicated an enrichment in the up-regulated transcripts and proteins involved in metabolic processes, especially those related to rRNA and ncRNA processing. Conversely, transcripts and proteins that were down-regulated showed significant clusters in catabolic processes, extracellular matrix, and cytoskeleton organization as well as in nephron and glomerulus development. Conclusion Using our zebrafish model, in-depth transcriptomics and proteomics analysis provided novel insights into the pathogenesis of podocyte-associated glomerular diseases. Data suggest, that pathological changes are accompanied by alternative splice events which might therefore be a new therapeutic approach in glomerular nephropathy.

Quantifying Variability in Zebrafish Larvae Locomotor Behavior across Experimental Conditions: A Learning-Based Tracker

This study investigated the effects of environmental changes on zebrafish larval behavior, using single-factor and orthogonal experiments to assess locomotion during temperature and pH changes. In single-factor experiments, zebrafish larvae were exposed to variations in temperature (22 to 30 °C) and pH levels (6.0, 7.0, 9.0). The simultaneous temperature and pH changes were investigated by orthogonal tests. In both experiments, each zebrafish larva was recorded in three 5 min videos at different stages (before exposure, during short-term exposure (10 min), and after long-term exposure (60 min)). You Look Only Once (YOLOv5) and Deep Simple Online Real Time Tracking (DeepSORT) models were adopted to develop a zebrafish larva tracking system, and YOLOv5 was improved in two aspects of anchor clustering and network structure. The tracking accuracy of the tracking system for small targets effectively improved, reaching more than 98% MOTA (Multiple Object Tracking Accuracy). Principal Component Analysis (PCA) was employed to extract three behavioral features from 13 motion parameters, namely motion activity, edge behavior, and motion direction preference. Our findings reveal that lower temperatures and acidic conditions both led to a decrease in motion behavioral activity, and the former also increased edge behavior. Conversely, elevated temperatures and alkaline conditions had a muted impact on these behaviors. Interestingly, concurrent changes in temperature and pH significantly altered directional preference. Additionally, we observed that lower temperatures elicited distinct temporal behavioral patterns at a constant pH level. In summary, we recommend the precise control and explicit reporting of ambient temperature and pH in both breeding devices and experimental wells to minimize the environmental impact on zebrafish behavior and enhance experiment repeatability and reliability.

#2641 MiR-200b induces ZEB1 mediated upregulation of Matriptase and affects expression of podocin

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and a predominant reason for renal failure. Evidence from previous research indicates that abnormal expression of microRNAs (miRs) links closely to the occurrence and progression of DKD. Methods Expression of miRs in urine samples from DKD patients and in cultured renal cells stressed with transforming growth factor beta 1 (TGF-β1) was evaluated by TaqMan assay. The emerging miR of interest and its downstream targets were analyzed in human renal sections, in cultured human podocytes and in zebrafish. Results Cell free urine screening showed increased abundance of miR-200b in samples from patients with DKD when compared to healthy controls. In situ hybridization confirmed expression of miR-200b in kidney specimens from DKD patients, but was not detected in healthy renal parenchyma. Transcriptome profiling of cultured human podocytes overexpressing miR-200b identified this miR as a regulator of the ZEB1 mediated matriptase (ST14)/ podocin pathway and found TGF- β1 stress to be upstream activators of miR-200b expression in these cells. Immunofluorescence analysis of kidney sections from DKD patients showed increased expression of ST14 and decreased expression of podocin when compared to healthy controls. Furthermore, injection of a miR-200b mimic in zebrafish larvae caused an upregulation of ST14 and was accompanied by phenotypic changes such as edema, proteinuria and podocyte effacement. Conclusion Our results show that miR-200b is expressed in glomeruli and found at elevated levels in urine of DKD patients. Overexpression of miR-200b in cultured podocytes and in zebrafish induces upregulation of matriptase, a serine protease previously shown to cleave podocin. The observed increase in matriptase and decrease in podocin levels in glomeruli from DKD patients, together with the effect of a miR-200b mimic on proteinuria and ultrastructure of the pronephros in zebrafish positions miR-200b as a key regulator that modulates matriptase overactivation in DKD and warrants further studies.

Norepinephrine changes behavioral state via astroglial purinergic signaling.

Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.

Ciliogenesis defects after neurulation impact brain development and neuronal activity in larval zebrafish

Cilia are slender, hair-like structures extending from cell surfaces and playing essential roles in diverse physiological processes. Within the nervous system, primary cilia contribute to signaling and sensory perception, while motile cilia facilitate cerebrospinal fluid flow. Here, we investigated the impact of ciliary loss on neural circuit development using a zebrafish line displaying ciliogenesis defects. We found that cilia defects after neurulation affect neurogenesis and brain morphology, especially in the cerebellum, and lead to altered gene expression profiles. Using whole brain calcium imaging, we measured reduced light-evoked and spontaneous neuronal activity in all brain regions. By shedding light on the intricate role of cilia in neural circuit formation and function in the zebrafish, our work highlights their evolutionary conserved role in the brain and sets the stage for future analysis of ciliopathy models.

Aluminum chloride and D-galactose induced a zebrafish model of Alzheimer's disease with cognitive deficits and aging

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder. Transgenic and pharmacological AD models are extensively studied to understand AD mechanisms and drug discovery. However, they are time-consuming and relatively costly, which hinders the discovery of potential anti-AD therapeutics. Here, we established a new model of AD in larval zebrafish by co-treatment with aluminum chloride (AlCl3) and D-galactose (D-gal) for 72 h. In particular, exposure to 150 μM AlCl3 + 40 mg/mL D-gal, 200 μM AlCl3 + 30 mg/mL D-gal, or 200 μM AlCl3 + 40 mg/mL D-gal successfully induced AD-like symptoms and aging features. Co-treatment with AlCl3 and D-gal caused significant learning and memory deficits, as well as impaired response ability and locomotor capacity in the plus-maze and light/dark test. Moreover, increased acetylcholinesterase and β-galactosidase activities, β-amyloid 1–42 deposition, reduced telomerase activity, elevated interleukin 1 beta mRNA expression, and enhanced reactive oxygen species production were also observed. In conclusion, our zebrafish model is simple, rapid, effective and affordable, incorporating key features of AD and aging, thus may become a unique and powerful tool for high-throughput screening of anti-AD compounds in vivo.

Norepinephrine Signals Through Astrocytes To Modulate Synapses.

Locus coeruleus (LC)-derived norepinephrine (NE) drives network and behavioral adaptations to environmental saliencies by reconfiguring circuit connectivity, but the underlying synapse-level mechanisms are elusive. Here, we show that NE remodeling of synaptic function is independent from its binding on neuronal receptors. Instead, astrocytic adrenergic receptors and Ca2+ dynamics fully gate the effect of NE on synapses as the astrocyte-specific deletion of adrenergic receptors and three independent astrocyte-silencing approaches all render synapses insensitive to NE. Additionally, we find that NE suppression of synaptic strength results from an ATP-derived and adenosine A1 receptor-mediated control of presynaptic efficacy. An accompanying study from Chen et al. reveals the existence of an analogous pathway in the larval zebrafish and highlights its importance to behavioral state transitions. Together, these findings fuel a new model wherein astrocytes are a core component of neuromodulatory systems and the circuit effector through which norepinephrine produces network and behavioral adaptations, challenging an 80-year-old status quo.

Vertebrates on a Chip: Noninvasive Electrical and Optical Mapping of Whole Brain Activity Associated with Pharmacological Treatments.

Mapping brain activities is necessary for understanding brain physiology and discovering new treatments for neurological disorders. Such efforts have greatly benefited from the advancement in technologies for analyzing neural activity with improving temporal or spatial resolution. Here, we constructed a multielectrode array based brain activity mapping (BAM) system capable of stabilizing and orienting zebrafish larvae for recording electroencephalogram (EEG) like local field potential (LFP) signals and brain-wide calcium dynamics in awake zebrafish. Particularly, we designed a zebrafish trap chip that integrates with an eight-by-eight surface electrode array, so that brain electrophysiology can be noninvasively recorded in an agarose-free and anesthetic-free format with a high temporal resolution of 40 μs, matching the capability typically achieved by invasive LFP recording. Benefiting from the specially designed hybrid system, we can also conduct calcium imaging directly on immobilized awake larval zebrafish, which further supplies us with high spatial resolution brain-wide activity data. All of these innovations reconcile the limitations of sole LFP recording or calcium imaging, emphasizing a synergy of combining electrical and optical modalities within one unified device for activity mapping across a whole vertebrate brain with both improved spatial and temporal resolutions. The compatibility with in vivo drug treatment further makes it suitable for pharmacology studies based on multimodal measurement of brain-wide physiology.