Zebrafish Locomotor Research Articles

Deep learning-assisted detection of psychoactive water pollutants using behavioral profiling of zebrafish embryos

Water pollution poses a significant risk to the environment and human health, necessitating the development of innovative detection methods. In this study, a series of representative psychoactive compounds were selected as model pollutants, and a new approach combining zebrafish embryo behavioral phenotyping with deep learning was used to identify and classify water pollutants. Zebrafish embryos were exposed to 17 psychoactive compounds at environmentally relevant concentrations (1 and 10 μg/L), and their locomotor behavior was recorded at 5 and 6 days post-fertilization (dpf). We constructed six distinct zebrafish locomotor track datasets encompassing various observation times and developmental stages and evaluated multiple deep learning models on these datasets. The results demonstrated that the ResNet101 model performed optimally on the 1-min track dataset at 6 dpf, achieving an accuracy of 65.35 %. Interpretability analyses revealed that the model effectively focused on the relevant locomotor track features for classification. These findings suggest that the integration of zebrafish embryo behavioral analysis with deep learning can serve as an environmentally friendly and economical method for detecting water pollutants. This approach offers a new perspective for water quality monitoring and has the potential to assist existing chemical analytical techniques in detection, thereby advancing environmental toxicology research and water pollution control efforts.

Fish 3D Locomotion app: a user-friendly computer application package for automatic data calculation and endpoint extraction for novel tank behavior in fish.

This paper introduces the Fish 3D Locomotion app (F3LA), a Python-based, Graphical User Interface (GUI)-equipped tool designed to automate behavioral endpoint extraction in zebrafish locomotion assays. Building on our previous work, which utilized a specialized aquatic tank with a mirror and a single camera for fish movement tracking in three dimensions, F3LA significantly enhances data processing efficiency. Its accuracy was tested by reanalyzing and comprehensively comparing the calculated data with the previously published data from prior publications. From the comparison results, 90% of endpoints showed a similar statistical difference result. These minor differences were due to the different starting points for the dataset and updated calculation formulas that are implemented in F3LA. In addition, shoaling area or shoaling volume calculations are also included in F3LA as a new feature that can serve as sensitive indicators of social cohesion, group dynamics, or stress responses, offering insights into neuropsychological conditions or the effects of pharmacological interventions. Furthermore, F3LA offers a marked improvement over manual operations, being at least five times faster, while maintaining consistent accuracy as it reduces human-induced errors, ensuring a higher degree of reliability in the results. Finally, the potency of F3LA was tested to evaluate the toxicities of 14 rare earth elements (REEs) to the adult zebrafish behaviors. Based on the results, our findings suggested that each tested REE altered fish behaviors in different patterns and magnitudes to each other. However, among the tested light rare earth elements (LREEs), neodymium was demonstrated to cause more relatively severe behavior alterations than other LREEs, indicated by the statistically higher value of entropy (0.2695 ± 0.04977 (mean with a standard deviation)) than the control group (0.2352 ± 0.05896). Meanwhile, in terms of heavy rare earth elements (HREEs), erbium seemed to lead to more distinct behavior toxicities than other HREEs, which was shown by the statistically lower level of fractal dimension (2.022 ± 0.3412) than the untreated group (2.255 ± 0.1661). Taken together, F3LA's development marks a significant advance in high-throughput toxicological and pharmacological assessments in zebrafish, leveraging three-dimensional locomotion data for a more comprehensive analysis of fish behavior performance, providing a significant contribution to research in various fields.

Effect of polar fractions of Marsilea crenata C. Presl. leaves in zebrafish locomotor activity.

Neurodegenerative diseases (NDs) are pathological conditions initiated by the loss of neuronal cell structure and the progressive decline in function caused by prolonged neuroinflammation. Postmenopausal women are at a high risk of experiencing NDs due to estrogen deficiency in their bodies, necessitating the administration of phytoestrogens as a replacement for estrogen in the body. One alternative therapy is administering phytoestrogens, estrogen-like substances from plants, which can be obtained from Marsilea crenata C. Presl. leaves. The purpose of this study was to determine whether administration of the n-butanol fraction (BF) and water fraction (WF) of M. crenata leaves could increase locomotor activity in rotenone-induced zebrafish. Treatment was given to each group of zebrafish with BF and WF at doses of 2.5; 5; 10; and 20 ppm to determine the locomotor activity. Then an analysis was carried out by looking at each movement of the zebrafish swimming for 1 min at the time of observation on days 0, 7, 14, 21, and 28. The result showed that BF and WF significantly increased the locomotor activity of zebrafish at the optimum dose of 20 ppm for BF and 5 ppm for WF compared to the negative control. This concludes that the polar fraction of M. crenata leaves is proven to have the potential to prevent ND progressivity.

Evaluation of Rhodanine Indolinones as AANAT Inhibitors.

Circadian rhythm (CR) dysregulation negatively impacts health and contributes to mental disorders. The role of melatonin, a hormone intricately linked to CR, is still a subject of active study. The enzyme arylalkylamine N-acetyltransferase (AANAT) is responsible for melatonin synthesis, and it is a potential target for disorders that involve abnormally high melatonin levels, such as seasonal affective disorder (SAD). Current AANAT inhibitors suffer from poor cell permeability, selectivity, and/or potency. To address the latter, we have employed an X-ray crystal-based model to guide the modification of a previously described AANAT inhibitor, containing a rhodanine-indolinone core. We made various structural modifications to the core structure, including testing the importance of a carboxylic acid group thought to bind in the CoA site, and we evaluated these changes using MD simulations in conjunction with enzymatic assay data. Additionally, we tested three AANAT inhibitors in a zebrafish locomotion model to determine their effects in vivo. Key discoveries were that potency could be modestly improved by replacing a 5-carbon alkyl chain with rings and that the central rhodanine ring could be replaced by other heterocycles and maintain potency.

Neural System Impairment and Involved Microglia-Neuron Regulation of Broflanilide in Zebrafish Larvae.

Broflanilide is widely used to control pests and has attracted attention due to its adverse effects on aquatic organisms. Our previous study showed that broflanilide has a negative impact on the central nervous system (CNS) at lethal dosages; however, its neural effects under practical situations and the underlying mechanisms remain unknown. To elucidate how broflanilide affects the CNS, we exposed zebrafish larvae to broflanilide at 16.9 and 88.0 μg/L (the environmentally relevant concentrations) for 120 h. Zebrafish locomotion was significantly disturbed at 88.0 μg/L, with a decreased moving distance and velocity accompanied by an inhibited neurotransmitter level. In vivo neuroimaging analysis indicated that the nerves of zebrafish larvae, including the axons, myelin sheaths, and neurons, were impaired. The number of neurons was significantly reduced after exposure, with an impaired morphological structure. These changes were accompanied by the abnormal transcription of genes involved in early CNS development. In addition, an increased total number of microglia and an elevated proportion of amoeboid microglia were observed after 88.0 μg/L broflanilide exposure, pointing out to an upstream role of microglia activation in mediating broflanilide neurotoxicity. Meanwhile, increased inflammatory cytokine levels and brain neutrophil numbers were observed, implicating significant inflammatory response and immune toxicity. Our findings indicate that broflanilide interferes with microglia-neuron regulation and induces neurodevelopmental disorders.

Combined effects of lead and manganese on locomotor activity and microbiota in zebrafish

Exposure to lead (Pb) and manganese (Mn) during early life influences neurodevelopment and increases the risk of neurodegenerative disorders. However, the level of developmental neurotoxicity due to combined exposure to the two metals remains unclear. Although the microbiota plays an essential part in the development of the nervous system via the gut–brain axis, there is a paucity of information regarding the interactions between exposure to Pb and Mn, the destruction of the microbiome, and neurodevelopmental impacts. To fill in this knowledge gap, we investigated the developmental neurotoxicity and effects on the microbiota of Pb (0.05 mg·L−1) alone and in combination with Mn (0.3 mg·L−1) in zebrafish larvae. Our results revealed that combined exposure precipitated higher malformation rates and lower locomotor activity levels than exposure to either Pb or Mn alone. Additionally, when we separated the combined exposure group from the other groups by applying unsupervised principal coordinates analysis (PCoA) and linear discriminant analysis (LEfSe) of microflora sequencing results, we observed extensive alterations in microbial abundances under combined-exposure conditions. Functional prediction analysis showed that combined exposure contributed to altered amino acid and lipid metabolism, and also that combined exposure to Pb and Mn reflected the greatest number of differentially activated biological pathways compared to the other three groups. ATP-binding cassette G (ABCG) genes and genes related to serotonin signaling and metabolism were altered following combined Pb and Mn exposure and exhibited disparate trends vis-à-vis Pb or Mn exposure alone. According to the results, the combined exposure to Pb and Mn led to more severe effects on both zebrafish locomotor activity and gut microbial composition. We suggest that the microbiota contributes to the combined neurotoxicity by increasing ABCG5 and ABCG8 gene expression.

Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 μM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 μM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.

Keluwih (Artocarpus camansi) extract effects in zebrafish models of Parkinson’s disease

Parkinson’s disease is a condition that affects the central nervous system in the brain and is brought on by a lack of dopamine. Uncontrollable tremors, uncoordinated movement, and stiffness characterize Parkinson’s disease. Until now, the medication for Parkinson’s disease is limited to relieve the symptoms and maintain the quality of life; thus, the progression of the disease can be delayed. In order to search for alternative therapy from herbs, Kluwih (Artocarpus camansi) has been used traditionally to relieve convulsants. This research aims to observe 96% ethanol extract of A. camansi leaves in dopamine and locomotor activity in adult male and female zebrafish (Dario rerio). The A. camansi extract concentration was 2.5; 5; 7.5; and 10 mg/ml for 28 days. Zebrafish locomotion was observed for 5 minutes on days 0; 7; 14; 21; and 28. ELISA measured the observations of dopamine after 28 days. The 96% ethanol extract of A. camansi leaves at 5 mg/ml can increase dopamine levels after induced with rotenone, but the dopamine level decreased at 7,5 and 10 mg/ml. The maximal concentration to increase locomotor activity is also at 5 mg/ml, along with dopamine concentration. Our findings revealed that 5 mg/ml of 96% ethanol extract of A. camansi leaves was the optimal dosage to stimulate dopamine release and enhance locomotor activity.

Behavioral profile of adult zebrafish acutely exposed to a selective dopamine uptake inhibitor, GBR 12909.

The dopamine transporter (DAT) is the main regulator of dopamine concentration in the extrasynaptic space. The pharmacological inhibition of the DAT results in a wide spectrum of behavioral manifestations, which have been identified so far in a limited number of species, mostly in rodents. Here, we used another well-recognized model organism, the zebrafish (Danio rerio), to explore the behavioral effects of GBR 12909, a highly-affine selective DAT blocker. We evaluated zebrafish locomotion, novelty-related exploration, spatial cognition, and social phenotypes in the novel tank, habituation and shoaling tests, following acute 20-min water immersion in GBR 12909. Our findings show hypolocomotion, anxiety-like state, and impaired spatial cognition in fish acutely treated with GBR 12909. This behavioral profile generally parallels that of the DAT knockout rodents and zebrafish, and it overlaps with behavioral effects of other DAT-inhibiting drugs of abuse, such as cocaine and D-amphetamine. Collectively, our data support the utility of zebrafish in translational studies on DAT targeting neuropharmacology and strongly implicate DAT aberration as an important mechanisms involved in neurological and psychiatric diseases.

Neurodevelopmental toxicity of organophosphate flame retardant triphenyl phosphate (TPhP) on zebrafish (Danio rerio) at different life stages

As a substitute for polybrominated diphenyl ethers (PBDEs), organophosphate flame retardant triphenyl phosphate (TPhP) is widely used in our daily products and diffusely exists in our living surroundings, but there is a paucity of information concerning its neurodevelopmental toxicity. Herein, we investigated the effects of TPhP exposure on developmental parameters, locomotor behavior, oxidative stress, apoptosis and transcriptional levels in zebrafish at different developmental stages, so as to explore the effects of TPhP exposure on zebrafish neural development and the underlying molecular mechanisms. TPhP concentration gradient exposure reduced the survival rate, hatchability, heart rate, body length and eye distance of zebrafish embryos/larvae, and caused malformations of zebrafish larvae. TPhP also leads to abnormal locomotor behaviors, such as reduced swimming distance and swimming speed, and impaired panic avoidance reflex to high light stimulation. TPhP caused ROS accumulation in 96 hpf larvae and induced Nrf2-antioxidant response in zebrafish. In addition, TPhP further activated mitochondrial signaling pathways, which affected apoptosis in the zebrafish eye region, resulting in visual impairment. Neurodevelopmental (mbpa, syn2a, foxo3a and pax6a), Retinoid acid metabolism (cyp26a1, raraa, rbp5, rdh1, crabp1a and rbp2a) and apoptosis-related genes (bcl2a, baxa and casp9) revealed the molecular mechanism of abnormal behavior and phenotypic symptoms, and also indicated that 96 hpf larvae are more sensitive than 7 dpf larvae. Thus, in the present study, we revealed the neurotoxic effects of TPhP at different early life stages in zebrafish, and zebrafish locomotor behavior impairments induced by TPhP exposure are attributed to co-regulation of visuomotor dysfunction and neuro-related genes. These results suggest that the safety of TPhP in organisms and even in humans needs to be further studied.

Pharmacodynamic Material Basis and Potential Mechanism Study of Spatholobi Caulis in Reversing Osteoporosis.

To elucidate the mechanism of Spatholobi Caulis (SC) in treating osteoporosis (OP) integrated zebrafish model and bioinformatics. Skeleton staining coupled with image quantification was performed to evaluate the effects of SC on skeleton mineralization area (SSA) and total optical density (TOD). Zebrafish locomotor activity was monitored using the EthoVision XT. Bioactive compounds of SC and their corresponding protein targets were acquired from Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Potential therapeutic targets for OP were summarized through retrieving 5 databases, and then, the overlapping genes between SC and OP were acquired. The core genes were selected by CytoHubba. Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) functional analysis of the intersection target genes were carried out by R software. Finally, the molecular docking simulation was manipulated between the ingredients and the hub genes. Compared with the model group, SC significantly increased the SSA and TOD at 10 mg/mL and improved the locomotor activity in a dose-dependent manner (p < 0.001). 33 components of SC were associated with 72 OP-related genes including 10 core genes (MAPK1, VEGFA, MMP9, AKT1, AR, IL6, CALM3, TP53, EGFR, and CAT). Advanced Glycation End Product (AGE) Receptor for AGE (RAGE) signaling pathway was screened out as the principal pathway of SC in anti-OP. The bioactive components (Aloe-emodin, Emodin, Formononetin, Licochalcone A, Luteolin, and Lopac-I-3766) have excellent affinity to core genes (MAPK1, VEGFA, MMP9, AKT1, and IL6). SC had the hierarchical network characteristics of "multicomponents/multitargets/multifunctions/multipathways" in reversing OP, but AGE-RAGE signaling pathway may be the main regulatory mechanism.

The Effect of Ethanol Extract of Marsilea crenata Presley Leaves on Rotenone-Induced Zebrafish Locomotor Activity

Neurodegenerative diseases are mostly experienced by postmenopausal women and are often caused by estrogen deficiency, so it is necessary to replace the hormone estrogen in the form of phytoestrogens. One of the plants that contain phytoestrogens is Marsilea crenata. The objective of this study is to see if giving rotenone-induced zebrafish a 96% ethanol extract of M. crenata leaves increases locomotor activity. This research was conducted by inducing 5 µg/L rotenone as a compound that will interfere with the locomotor activity of zebrafish. Next, treatment was given with 96% ethanol extract of clover in each group with a dose of 2.5; 5; 10; and 20 mg/mL to determine the effect of the extract on increasing locomotor activity of rotenone-induced zebrafish. Observations were made by looking at the quantity of zebrafish swimming every five minutes until day 28. The treatment of 96% ethanol extract of M. crenata leaves could significantly increase zebrafish motility at the optimum dose of 2.5 mg/mL, because every week the zebrafish locomotor activity increased compared to the negative control. M. crenata leaves extract is proven to prevent neurodegenerative diseases. However, further research needs to be done on the degenerative effects of rotenone every week.

Parkia platycephala Lectin (PPL) Inhibits Orofacial Nociception Responses via TRPV1 Modulation.

Lectins are a heterogeneous group of proteins that reversibly bind to simple sugars or complex carbohydrates. The plant lectin purified from the seed of Parkia platycephala (PPL) was studied. This study aimed to investigate the possible orofacial antinociceptive of PPL lectin in adult zebrafish and rodents. Acute nociception was induced by cinnamaldehyde (0.66 μg/mL), 0.1% acidified saline, glutamate (12.5 µM) or hypertonic saline (5 M NaCl) applied into the upper lip (5.0 µL) of adult wild zebrafish. Zebrafish were pretreated by intraperitoneal injection (20 µL) with vehicle (Control) or PPL (0.025; 0.05 or 0.1 mg/mL) 30 min before induction. The effect of PPL on zebrafish locomotor behaviour was evaluated in the open field test. Naive groups were included in all tests. In one experiment, animals were pre-treated with capsazepine to investigate the mechanism of antinociception. The involvement of central afferent C-fibres was also investigated. In another experiment, rats pre-treated with PPL or saline were submitted to the temporomandibular joint formalin test. Other groups of rats were submitted to infraorbital nerve transection to induce chronic pain, followed by induction of mechanical sensitivity using von Frey. PPL reduced nociceptive behaviour in adult zebrafish, and this is related to the activation of the TRPV1 channels since antinociception was effectively inhibited by capsazepine and by capsaicin-induced desensitization. PPL reduced nociceptive behaviour associated with temporomandibular joint and neuropathic pain. The results confirm the potential pharmacological relevance of PPL as an inhibitor of orofacial nociception in acute and chronic pain.

Effects of super-class cannabis terpenes beta-caryophyllene and alpha-pinene on zebrafish behavioural biomarkers

Terpenes possess a wide range of medicinal properties and are potential therapeutics for a variety of pathological conditions. This study investigated the acute effects of two cannabis terpenes, β-caryophyllene and α-pinene, on zebrafish locomotion, anxiety-like, and boldness behaviour using the open field exploration and novel object approach tests. β-caryophyllene was administered in 0.02%, 0.2%, 2.0%, and 4% doses. α-pinene was administered in 0.01%, 0.02%, and 0.1% doses. As α-pinene is a racemic compound, we also tested its (+) and (−) enantiomers to observe any differential effects. β-caryophyllene had only a sedative effect at the highest dose tested. α-pinene had differing dose-dependent effects on anxiety-like and motor variables. Specifically, (+)-α-pinene and (−)-α-pinene had significant effects on anxiety measures, time spent in the thigmotaxis (outer) or center zone, in the open field test, as well as locomotor variables, swimming velocity and immobility. (+ /−)-α-pinene showed only a small effect on the open field test on immobility at the 0.1% dose. This study demonstrates that α-pinene can have a sedative or anxiolytic effect in zebrafish and may have different medicinal properties when isolated into its (+) or (−) enantiomers.

ВЛИЯНИЕ МАГНИТНЫХ ФЛУКТУАЦИЙ НА ЦИРКАДНУЮ СТРУКТУРУ ДВИГАТЕЛЬНОЙ АКТИВНОСТИ У DANIO RERIO (ПРЕДВАРИТЕЛЬНЫЕ ДАННЫЕ)

The locomotor activity of zebrafish (Danio rerio) has a pronounced, well-studied circadian rhythm. Under constant illumination, the period of free-running locomotor activity in this species usually becomes less than 24 hours. To evaluate the entraining capabilities of slow magnetic variations, zebrafish locomotor activity was evaluated at constant illumination and fluctuating magnetic field with a period of 26.8 hours. Lomb-Scargle periodogram revealed significant free-running rhythms of locomotor activity and related behavioral endpoints with a period close to 27 hours. Obtained results reveal the potential of slow magnetic fluctuations for entrainment of the circadian rhythms in zebrafish. The putative mechanisms responsible for the entrainment are discussed, including the possible role of cryptochromes.

Structural and spectroscopic analysis, ADMET study, and anxiolytic-like effect in adult zebrafish (Danio rerio) of 4′-[(1E,2E)-1-(2-(2′,4′-dinitrophenyl)hydrazone-3-(4-methoxyphenyl)allyl)aniline

Benzodiazepines (GABA receptor agonists) and serotonin reuptake inhibitors (SSRIs) are the drugs of choice for the treatment of anxiety. However, they have considerable adverse effects, so it is important to discover new anxiolytic agents. In this work, the 4′-[(1E,2E)-1-(2-(2′,4′-dinitrophenyl)hydrazone-3-(4-methoxyphenyl)allyl)aniline (HYDRAZDN) was synthesized and characterized by 1H and 13C NMR, 2D-NMR, ATR-FTIR, FT-Raman, and UV–Vis spectroscopy, and their structural and spectroscopic proprieties were evaluated by quantum chemistry theoretical calculations. Properties such as drug-likeness and pharmacokinetics were also estimated. The anxiolytic-like effect of the compound was also evaluated using adult zebrafish (Danio rerio). The results showed that the HYDRAZDN compound caused a reduction in zebrafish locomotor activity and induced maximum permanence of zebrafish in the clear zone at a dose of 40 mg/kg, an effect similar to the effect of positive control diazepam (DZP: 40 mg/kg). To identify the mechanism of action of the anxiolytic effect of this compound, we used flumazenil, an antagonist at the benzodiazepine binding site at GABAA receptors. The treatment with the highest dose of HYDRAZDN (40 mg/kg) and DZP (40 mg/kg) caused anxiolytic behavior in the animals that spent most of their time in the dark region of the aquarium. This effect was reduced by flumazenil, indicating that anxiolytic activity of the synthesized hydrazone occurs through the GABAergic system.

Mathematical modeling of zebrafish social behavior in response to acute caffeine administration.

Zebrafish is a model organism that is receiving considerable attention in preclinical research. Particularly important is the use of zebrafish in behavioral pharmacology, where a number of high-throughput experimental paradigms have been proposed to quantify the effect of psychoactive substances consequences on individual and social behavior. In an effort to assist experimental research and improve animal welfare, we propose a mathematical model for the social behavior of groups of zebrafish swimming in a shallow water tank in response to the administration of psychoactive compounds to select individuals. We specialize the mathematical model to caffeine, a popular anxiogenic compound. Each fish is assigned to a Markov chain that describes transitions between freezing and swimming. When swimming, zebrafish locomotion is modeled as a pair of coupled stochastic differential equations, describing the time evolution of the turn-rate and speed in response to caffeine administration. Comparison with experimental results demonstrates the accuracy of the model and its potential use in the design of in-silico experiments.

Selective actions of benzodiazepines at the transmembrane anaesthetic binding sites of the GABAA receptor: In vitro and in vivo studies.

In addition to binding to the classical high-affinity extracellular benzodiazepine binding site of the GABAA receptor, some benzodiazepines occupy transmembrane inter-subunit anaesthetic sites that bind etomidate (β+ /α- sites) or the barbiturate derivative R-mTFD-MPAB (α+ /β- and γ+ /β- sites). We aimed to define the functional effects of these interactions on GABAA receptor activity and animal behaviour. With flumazenil blocking classical high-affinity extracellular benzodiazepine site effects, modulation of GABA-activated currents by diazepam, midazolam and flurazepam was measured electrophysiologically in wildtype and M2-15' mutant α1 β3 γ2L GABAA receptors. Zebrafish locomotive activity was also assessed in the presence of each benzodiazepine plus flumazenil. In the presence of flumazenil, micromolar concentrations of diazepam and midazolam both potentiated and inhibited wildtype GABAA receptor currents. β3 N265M (M2-15' in the β+ /α- sites) and α1 S270I (M2-15' in the α+ /β- site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ2 S280W mutation (M2-15' in the γ+ /β- site) abolished inhibition. Flurazepam plus flumazenil only inhibited wildtype receptor currents, an effect unaltered by M2-15' mutations. In the presence of flumazenil, zebrafish locomotion was enhanced by diazepam at concentrations up to 30 μM and suppressed at 100 μM, suppressed by midazolam and enhanced by flurazepam. Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.

Zebrafish larva’s response and habituation to electric signal: Effects of voltage, current and pulsation studied in a microfluidic device

We previously showed that electric current induces zebrafish larvae to move towards the anode pole along a microchannel. For a larva with a fixed head and a moving tail, we observed that the response to electricity depended on the current magnitude. The effects of electric signal direction, voltage magnitude and habituation to repeated exposures to electric pulses were not characterized. Here, this knowledge gap was addressed by exploiting them in a microfluidic device with a head-trap to immobilize a zebrafish larva and a downstream chamber for tail movement phenotypic characterization based on response duration (RD) and tail beat frequency (TBF). We first assessed larvae’s response to electric current direction (at 3 µA) and voltage magnitude. Changing the current direction significantly altered the RD and TBF with long and low-frequency responses seen when the anode was positioned at the larvae’s tail. The electric voltage had a significant effect on larvae’s locomotion with long RD and low TBF observed at 5.6 V in the range of 1.3–9 V. We also demonstrated that the zebrafish locomotor response to repeated 3 µA current pulses diminished with dependency on the interstimulus interval. However, the diminished response was fully recovered after a 5-min resting period or introduction of a novel light stimulus (i.e., habituation-dishabituation strategy). Therefore, electric response suppression in zebrafish was attributed to the habituation as a form of non-associative learning. Our microfluidic platform has a broad application potential in behavioral neuroscience to study cognitive phenotypes, fundamental studies on the biological roots of electric response, and in pharmacological screening.

Estrogen receptor: A potential linker of estrogenic and dopaminergic pathways in zebrafish larvae following deltamethrin exposure

Deltamethrin (DM), a type II pyrethroid insecticide, is widely used to control agricultural pests. However, its excessive use exerts a detrimental effect on the ecological environment and human health, indicating the need to study its potential risks in detail. In the present study, zebrafish embryos were exposed to varying concentrations of DM (0.1, 1, 10, and 25 μg/L) for 96 h to assess the alterations in the transcript levels of proteins of the estrogenic and dopaminergic pathways. In addition, its effect on zebrafish locomotor activity was studied. The mRNA expression of cyp19a1b, erα, erβ2, fshr, gnrh2, gnrhr3, vtg3, dat, and dr1 significantly changed after exposing the embryos to DM. Deltamethrin at 10 and 25 μg/L significantly reduced the average swimming speed of zebrafish larvae. In addition, embryos injected with zebrafish estrogen receptor α (erα) and β (erβ) morpholinos and co-exposed to 25 μg/L DM for 96 h showed reduced expression of vtg3 mRNA compared to embryos exposed to 25 μg/L DM only. The locomotor activity of erα and erβ knockdown zebrafish following DM exposure was increased significantly when compared with that of larvae exposed to 25 μg/L DM only. Our results demonstrated that DM altered the locomotor activity of zebrafish larvae and the transcript levels of the components of estrogenic and dopaminergic pathways; erα and erβ knockdown weakened these effects.