Locomotion Assays Research Articles

Toxicity of zinc oxide nanoparticles: Cellular and behavioural effects

Due to their extensive use, the release of zinc oxide nanoparticles (ZnO NP) into the environment is increasing and may lead to unintended risk to both human health and ecosystems. Access of ZnO NP to the brain has been demonstrated, so their potential toxicity on the nervous system is a matter of particular concern. Although evaluation of ZnO NP toxicity has been reported in several previous studies, the specific effects on the nervous system are not completely understood and, particularly, effects on genetic material and on organism behaviour are poorly addressed. We evaluated the potential toxic effects of ZnO NP in vitro and in vivo, and the role of zinc ions (Zn2+) in these effects. In vitro, the ability of ZnO NP to be internalized by A172 glial cells was verified, and the cytotoxic and genotoxic effects of ZnO NP or the released Zn2+ ions were addressed by means of vital dye exclusion and comet assay, respectively. In vivo, behavioural alterations were evaluated in zebrafish embryos using a total locomotion assay. ZnO NP induced decreases in viability of A172 cells after 24 h of exposure and genetic damage after 3 and 24 h. The involvement of the Zn2+ ions released from the NP in genotoxicity was confirmed. ZnO NP exposure also resulted in decreased locomotor activity of zebrafish embryos, with a clear role of released Zn2+ ions in this effect. These findings support the toxic potential of ZnO NP showing, for the first time, genetic effects on glial cells and proving the intervention of Zn2+ ions.

Structural insights into the molecular effects of the anthelmintics monepantel and betaine on the Caenorhabditis elegans acetylcholine receptor ACR-23

Anthelmintics are drugs used for controlling pathogenic helminths in animals and plants. The natural compound betaine and the recently developed synthetic compound monepantel are both anthelmintics that target the acetylcholine receptor ACR-23 and its homologs in nematodes. Here, we present cryo-electron microscopy structures of ACR-23 in apo, betaine-bound, and betaine- and monepantel-bound states. We show that ACR-23 forms a homo-pentameric channel, similar to some other pentameric ligand-gated ion channels (pLGICs). While betaine molecules are bound to the classical neurotransmitter sites in the inter-subunit interfaces in the extracellular domain, monepantel molecules are bound to allosteric sites formed in the inter-subunit interfaces in the transmembrane domain of the receptor. Although the pore remains closed in betaine-bound state, monepantel binding results in an open channel by wedging into the cleft between the transmembrane domains of two neighboring subunits, which causes dilation of the ion conduction pore. By combining structural analyses with site-directed mutagenesis, electrophysiology and in vivo locomotion assays, we provide insights into the mechanism of action of the anthelmintics monepantel and betaine.

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.

Optimization of Laser-Based Method to Conduct Skin Ablation in Zebrafish and Development of Deep Learning-Based Method for Skin Wound-Size Measurement

Skin plays an important role as a defense mechanism against environmental pathogens in organisms such as humans or animals. Once the skin integrity is disturbed by a wound, pathogens can penetrate easily into a deeper part of the body to induce disease. By this means, it is important for the skin to regenerate quickly upon injury to regain its protective barrier function. Traditionally, scientists use rodents or mammals as experimental animals to study skin wound healing. However, due to concerns about animal welfare and increasing costs of laboratory animals, such as rodents, scientists have considered alternative methods of implementing replace, reduce, and refine (3Rs) in experimentation. Moreover, several previous studies on skin wound healing in fish used relatively expensive medical-grade lasers with a low calculation efficiency of the wound area, which led to human judgment errors. Thus, this study aimed to develop a new alternative model for skin wound healing by utilizing zebrafish together with a new rapid and efficient method as an alternative in investigating skin wound healing. First, in order to fulfill the 3Rs concept, the pain in the tested zebrafish was evaluated by using a 3D locomotion assay. Afterward, the obtained behavior data were analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons tests; later, 3 watts was chosen as the power for the laser, since the wound caused by the laser at this power did not significantly alter zebrafish swimming behaviors. Furthermore, we also optimized the experimental conditions of zebrafish skin wound healing using a laser engraving machine, which can create skin wounds with a high reproducibility in size and depth. The wound closure of the tested zebrafish was then analyzed by using a two-way ANOVA, and presented in 25%, 50%, and 75% of wound-closure percentages. After imparting wounds to the skin of the zebrafish, wound images were collected and used for deep-learning training by convolutional neural networks (CNNs), either the Mask-RCNN or U-Net, so that the computer could calculate the area of the skin wounds in an automatic manner. Using ImageJ manual counting as a gold standard, we found that the U-Net performance was better than the Mask RCNN for zebrafish skin wound judgment. For proof-of-concept validation, a U-Net trained model was applied to study and determine the effect of different temperatures and the administration of antioxidants on the skin wound-healing kinetics. Results showed a significant positive correlation between the speed of wound closure and the exposure to different temperatures and administration of antioxidants. Taken together, the laser-based skin ablation and deep learning-based wound-size measurement methods reported in this study provide a faster, reliable, and reduced suffering protocol to conduct skin wound healing in zebrafish for the first time.

Adaptive tail-length evolution in deer mice is associated with differential Hoxd13 expression in early development

Variation in the size and number of axial segments underlies much of the diversity in animal body plans. Here we investigate the evolutionary, genetic and developmental mechanisms driving tail-length differences between forest and prairie ecotypes of deer mice (Peromyscus maniculatus). We first show that long-tailed forest mice perform better in an arboreal locomotion assay, consistent with tails being important for balance during climbing. We then identify six genomic regions that contribute to differences in tail length, three of which associate with caudal vertebra length and the other three with vertebra number. For all six loci, the forest allele increases tail length, indicative of the cumulative effect of natural selection. Two of the genomic regions associated with variation in vertebra number contain Hox gene clusters. Of those, we find an allele-specific decrease in Hoxd13 expression in the embryonic tail bud of long-tailed forest mice, consistent with its role in axial elongation. Additionally, we find that forest embryos have more presomitic mesoderm than prairie embryos and that this correlates with an increase in the number of neuromesodermal progenitors, which are modulated by Hox13 paralogues. Together, these results suggest a role for Hoxd13 in the development of natural variation in adaptive morphology on a microevolutionary timescale.

Combination nanochemotherapy of brain tumor using polymeric nanoparticles loaded with doxorubicin and paclitaxel: An in vitro and in vivo study

This study aims to overcome physiological barriers and increase the therapeutic index for the treatment of glioblastoma (GBM) tumors by using Paclitaxel (PTX) loaded Poly(lactic co-glycolic acid) nanoparticles (PTX-PLGA-NPs) and Doxorubicin (DOX) loaded Poly (lactic co-glycolic acid) nanoparticles (DOX-PLGA-NPs). The hydrodynamic diameter of nanoparticles (NPs) was characterized by dynamic light scattering (DLS) which was 94 ± 4 nm and 133 ± 6 nm for DOX-PLGA-NPs, and PTX-PLGA-NPs, respectively. The zeta potential for DOX-PLGA-NPs and PTX-PLGA-NPs were −15.2 ± 0.18 mV and −17.3 ± 0.34 mV, respectively. The cytotoxicity of PTX-PLGA-NPs and DOX-PLGA-NPs was augmented compared to DOX and PTX on C6 GBM cells. The Lactate dehydrogenase (LDH) tests for various formulations were carried out. The results indicated that the amount of released LDH was 262 ± 7.84 U.L-1 at the concentration of 2 mg/mL in the combination therapy, which was much higher than other groups (DOX-PLGA-NPs (210 ± 6.92 U.L-1), PTX-PLGA-NPs (201 ± 8.65 U.L-1), DOX (110 ± 9.81 U.L-1), PTX (95 ± 5.02 U.L-1) and PTX + DOX (67 ± 4.89 U.L-1)). MRI results of the combination therapy of PTX-PLGA-NPs and DOX-PLGA-NPs indicated that GBM tumor size decreased considerably compared to the other formulations. Also, combination therapy of PTX-PLGA-NPs and DOX-PLGA-NPs demonstrated a longer median survival of more than 80 days compared to PTX (38 days), DOX (37 days) and PTX + DOX (48 days), PTX-NPs (58 days) and DOX-NPs (62 days). The results of locomotion, body weight, rearing and grooming assays indicated that combination therapy of PTX-PLGA-NPs and DOX-PLGA-NPs had the most positive effect on the movements of rats compared to the other formulations.

Roscovitine’s Effect on D. melanogaster with TDP-43 Nuclear Loss Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurological disease that leads to motor neuron death, causing muscle atrophy and paralysis. The majority of ALS patients die from respiratory failure within 2–5 years. By 2040, the incidence of ALS is predicted to increase worldwide by 70%. ALS has no cure. TDP-43 protein dysfunction is present in ~97% of ALS patients. Past ALS research focused on TDP-43 aggregation in the cytoplasm of neuronal cells; however, loss of TDP-43 from the nucleus is now considered the main contributor to neurodegeneration. Drosophila larvae with dTDP-43 nuclear loss exhibit locomotion deficits and reduced levels of cacophony, a neuronal calcium channel required for neurotransmitter release. When cacophony was restored in dTDP-43 nuclear loss larvae, locomotion was rescued. Roscovitine is a drug that increases calcium influx in neuronal calcium channels, essentially performing the same function as increased cacophony. The purpose and novelty of this research are to determine if a roscovitine supplement can improve the locomotion of a TDP-43 nuclear loss ALS model of Drosophila melanogaster. The larval locomotion assay was used to validate the ALS symptom of muscle weakness. The movements of larvae on an agar plate were recorded. Using ImageJ, the displacements and speeds of the larvae were determined. Results indicate that ALS larvae fed roscovitine performed significantly better on the locomotion assay than ALS larvae fed normal food (p-value < 0.0001). This research provides insight into the role of neuronal calcium channels in TDP-43 nuclear loss and calcium channel agonists’ potential in treating ALS.

Acute administration of the NMDA receptor antagonists ketamine and MK-801 reveals dysregulation of glutamatergic signalling and sensorimotor gating in the Sapap3 knockout mouse model of compulsive-like behaviour

Obsessive-compulsive disorder (OCD) is characterised by excessive intrusive thoughts that may cause an individual to engage in compulsive behaviours. Frontline pharmacological treatments (i.e., selective serotonin reuptake inhibitors (SSRIs)) leave approximately 40% of patients refractory to treatment. To investigate the possibility of novel pharmacological therapies for OCD, as well as the potential mechanisms underlying its pathology, we used the Sapap3 knockout (KO) mouse model of OCD, which exhibits increased anxiety and compulsive grooming behaviours. Firstly, we investigated whether administration of the NMDA receptor (NMDAR) antagonist ketamine (30 mg/kg), would reduce anxiety and grooming behaviour in Sapap3 KO mice. Anxiety-like behaviour was measured via time spent in the light component of the light-dark box test. Grooming behaviour was recorded and scored in freely moving mice. In line with previous works conducted in older animals (i.e. typically between 6 and 9 months of age), we confirmed here that Sapap3 KO mice exhibit an anxious, compulsive grooming, hypolocomotive and reduced body weight phenotype even at a younger age (i.e., 2-3 months of age). However, we found that acute administration of ketamine did not cause a reduction in anxiety or grooming behaviour. We then investigated in vivo glutamatergic function via the administration of a different NMDAR antagonist, MK-801 (0.25 mg/kg), prior to locomotion and prepulse inhibition assays. We found evidence of altered functional NMDAR activity, as well as sexually dimorphic prepulse inhibition, a measure of sensorimotor gating, in Sapap3 KO mice. These results are suggestive of in vivo glutamatergic dysfunction and their functional consequences, enabling future research to further investigate novel treatments for OCD.

Local delivery of FK506 to a nerve allograft is comparable to systemic delivery at suppressing allogeneic graft rejection.

The objective of this study was to determine if locally delivered FK506 could prevent allogeneic nerve graft rejection long enough to allow axon regeneration to pass through the nerve graft. An 8mm mouse sciatic nerve gap injury repaired with a nerve allograft was used to assess the effectiveness of local FK506 immunosuppressive therapy. FK506-loaded poly(lactide-co-caprolactone) nerve conduits were used to provide sustained local FK506 delivery to nerve allografts. Continuous and temporary systemic FK506 therapy to nerve allografts, and autograft repair were used as control groups. Serial assessment of inflammatory cell and CD4+ cell infiltration into the nerve graft tissue was performed to characterize the immune response over time. Nerve regeneration and functional recovery was serially assessed by nerve histomorphometry, gastrocnemius muscle mass recovery, and the ladder rung skilled locomotion assay. At the end of the study, week 16, all the groups had similar levels of inflammatory cell infiltration. The local FK506 and continuous systemic FK506 groups had similar levels of CD4+ cell infiltration, however, it was significantly greater than the autograft control. In terms of nerve histmorphometry, the local FK506 and continunous systemic FK506 groups had similar amounts of myelinated axons, although they were significantly lower than the autograft and temporary systemic FK506 group. The autograft had significantly greater muscle mass recovery than all the other groups. In the ladder rung assay, the autograft, local FK506, and continuous systemic FK506 had similar levels of skilled locomotion performance, whereas the temporary systemic FK506 group had significanty better performance than all the other groups. The results of this study suggest that local delivery of FK506 can provide comparable immunosuppression and nerve regeneration outcomes as systemically delivered FK506.

Administration of Stephania tetrandra to Drosophila melanogaster to create obsessive compulsive disorder model

Obsessive compulsive disorder (OCD) is a neuropsychiatric condition that affects 2-3% of the world population, with more research needed to better understand its mechanisms. Theories positing the involvement of neurotransmitters like serotonin, dopamine, and glutamate in OCD have gained traction, however no specific cause has been identified. There are currently no research models of OCD in invertebrates, and existing vertebrate models fail to accurately model the disorder. Often, these existing models exhibit abnormal behaviors not associated with OCD, or do not respond appropriately to OCD treatments. The current study aimed to create a model of OCD in Drosophila melanogaster through administration of Stephania tetrandra extract. S. tetrandra extract is a plant extract with antiserotonergic and antidopaminergic properties similar to those of second-generation antipsychotics, a class of drugs that can induce obsessive compulsive symptoms (OCS). We hypothesized that flies administered the extract would display more OCS than control flies. We determined the highest non-lethal dose of S. tetrandra in Drosophila and administered it in two assays for OCS: a locomotion assay that measured movement as a metric of anxiety, and a grooming assay that quantified repetitive grooming behavior. These two assays were selected because OCD is characterized by repetitive behaviors and anxiety. Administration of the S. tetrandra extract significantly increased both anxiety and repetitive behavior, suggesting that flies administered the extract had increased OCS and that S. tetrandra treatment could serve as a potential model of OCD in Drosophila.

Ethylene thiourea exposure induces neurobehavioral toxicity in zebrafish by disrupting axon growth and neuromuscular junctions

Ethylene thiourea (ETU) converted from ethylene bisdithiocarbamate (EBDC) fungicides has aroused great concern because of its prevalence and harmful effects. Although ETU-induced neurotoxicity has been reported, the potential mechanisms remain unclear. This study provided insights into its neurotoxic effects at environmentally relevant concentrations in zebrafish. Our findings showed that embryonic exposure to ETU decreased the hatch rate and delayed somite development. Furthermore, ETU treatment significantly reduced the dark velocity in the locomotion assay. The upregulated tendency of the mitogen-activated protein kinases (MAPK) pathway (mknk1, atf4, mapkapk3) screened by transcriptome analysis implied motor neuron degeneration, which was validated by subsequent morphological observation, as axon length and branches were truncated in the 62.5 µg/L ETU group. However, although the rescue experiment with a p38 MAPK inhibitor (SB203580) successfully ameliorated axon degeneration, it failed to reverse the locomotion behaviors. Further exploration of transcriptome data revealed the varied expression of presynaptic scaffold protein-related genes (pcloa, pclob, bsna), whose downregulation might impair the neuromuscular junction (NMJ). Therefore, we reasonably suspected that ETU-induced neurobehavioral deficits might result from the combined effects of the MAPK pathway and presynaptic proteins. Considering this, we highlighted the necessity to take precautions and early interventions for susceptible ETU-exposed populations.

The light-independent locomotion response to a static magnetic field in Xenopus tadpoles

Increasing numbers of behavioral observations have shown that many organisms can sense magnetic fields in the environment. However, the underlying mechanisms of magnetosensation remain elusive, and a major problem is the lack of an animal model convenient for detecting magnetic responses, especially in vertebrates. We have previously reported that the physical performance of mice decreased after 1 month of exposure to a hypomagnetic field, which probably includes a light-independent mechanism. In this study, we investigated the effects of a static magnetic field on the locomotion of Xenopus tadpoles (stage 48) using a real-time recording system. Because it is speculated that the photosensing system is related to magnetosensation, we triggered the tadpoles to swim by a 30 s light pulse after a 5 min adaptation in the dark and measured the swimming activities of the tadpoles under static magnetic field exposure in both the dark and bright conditions. Compared to the sham control treatment, the presence of a magnet inhibited the movement of the tadpoles under both bright and dark conditions, as shown by reductions in swimming distance, speed, and counts of path adjustment. Moreover, the directional preference for path adaptation was altered when swimming in the dark. These results suggest that tadpoles in the dark can exert a rapid locomotion response to changes in the environmental magnetic field, providing evidence for an intrinsic, light-independent rapid magnetoresponse pathway in tadpoles. In addition, this rapid vertebrate locomotion assay paradigm will be a practical tool to facilitate further investigation of the mechanisms of biomagnetic effects.

The cGMP-dependent protein kinase gene can regulate trail-following behaviour and locomotion in the termite Reticulitermes chinensis Snyder.

Social behaviours in termites are closely related to the chemical communication between individuals. It is well known that foraging worker termites can use trail pheromones to orient and locomote along trails so as to take food resources back to the nest. However, it is still unclear how termites recognize trail pheromones. Here, we cloned and sequenced the cGMP-dependent protein kinase (PKG) gene from the termite Reticulitermes chinensis Snyder, and then examined the response of termites to trail pheromones after silencing PKG through RNA interference. We found that PKG knockdown impaired termite ability to follow trail pheromones accurately and exhibited irregular behavioural trajectories in response to the trail pheromone in the termite R. chinensis. Our locomotion assays further showed that PKG knockdown significantly increased the turn angle and angular velocity in the termite R. chinensis. These findings help us better understanding the molecular regulatory mechanism of foraging communications in termites.

FTD-associated mutations in Tau result in a combination of dominant and recessive phenotypes

Although mutations in the microtubules-associated protein Tau have long been connected with several neurodegenerative diseases, the underlying molecular mechanisms causing these tauopathies are still not fully understood. Studies in various models suggested that dominant gain-of-function effects underlie the pathogenicity of these mutants; however, there is also evidence that the loss of normal physiological functions of Tau plays a role in tauopathies. Previous studies on Tau in Drosophila involved expressing the human Tau protein in the background of the endogenous Tau gene in addition to inducing high expression levels. To study Tau pathology in more physiological conditions, we recently created Drosophila knock-in models that express either wildtype human Tau (hTauWT) or disease-associated mutant hTau (hTauV337M and hTauK369I) in place of the endogenous Drosophila Tau (dTau). Analyzing these flies as homozygotes, we could therefore detect recessive effects of the mutations while identifying dominant effects in heterozygotes. Using memory, locomotion and sleep assays, we found that homozygous mutant hTau flies showed deficits already when quite young whereas in heterozygous flies, disease phenotypes developed with aging. Homozygotes also revealed an increase in microtubule diameter, suggesting that changes in the cytoskeleton underlie the axonal degeneration we observed in these flies. In contrast, heterozygous mutant hTau flies showed abnormal axonal targeting and no detectable changes in microtubules. However, we previously showed that heterozygosity for hTauV337M interfered with synaptic homeostasis in central pacemaker neurons and we now show that heterozygous hTauK369I flies have decreased levels of proteins involved in the release of synaptic vesicles. Taken together, our results demonstrate that both mutations induce a combination of dominant and recessive disease-related phenotypes that provide behavioral and molecular insights into the etiology of Tauopathies.

Allosteric Dopamine transporter modulator inhibits cocaine‐induced behavior

The dopamine transporter (DAT) is a transmembrane protein responsible for reuptake the neurotransmitter dopamine back into the presynaptic neurons. Dopamine (DA) is involved in driving motivation, it is increased when people expect a reward, and it is also involved in many central nervous systems (CNS) disorders. The inhibition of the reuptake process prevents dopamine in the synaptic cleft from getting back into the presynaptic neuron. Cocaine and amphetamine both inhibit DA reuptake by binding to the orthosteric site but through different mechanisms. Cocaine is a classical inhibitor that blocks the transporter through competitive inhibition. On the other hand, amphetamine is a substrate of DAT and causes competitive inhibition of DA reuptake and reversal of the transporter leading to the release of DA. As a result of the DA reuptake inhibition, the DA in the synaptic cleft increases, leading to amplified downstream dopaminergic signaling primarily in the mesolimbic pathway. Because DAT is a primary target of psychostimulants, compounds acting on the DAT in novel ways could potentially treat psychostimulant use disorders. We previously found a novel compound—KM822 and characterized it as an allosteric modulator of DAT function that significantly decreases cocaine‐induced locomotive response in planarians. To test if KM822 has similar effects in mammals, we administered KM822 and cocaine through intracranial infusions into Long Evans rats' nucleus accumbens (NAc) and measured locomotion. We targeted the NAc as it is a part of the brain that plays a crucial role in the mesolimbic dopaminergic pathway and has been recognized by its high density of DAT. Our results showed that KM822 significantly decreased hyper‐locomotion induced by cocaine and notably did not cause any increase in locomotion by itself. KM822's ability to block hyper‐locomotion induced by amphetamine, another psychostimulant with a different mechanism of action, was also examined. Finally, we examined KM822's ability to interfere with cocaine's rewarding effect using the conditioned place preference (CPP) assay. In this assay, animals are tested for their preference for a cocaine‐associated environment. Unlike locomotion assay, this assay is more relevant to behaviors associated with cocaine addiction and therefore has high translational value. Overall, these studies demonstrated KM822's ability to block DAT inhibitor‐induced behaviors in rats and provided strong evidence that the novel allosteric DAT modulator KM822 has significant potential for treating psychostimulant addiction.

Effect of Water-Pipe Smoking on the Normal Development of Zebrafish.

Background: Among all types of tobacco consumption, Water-Pipe Smoking (WPS) is the most widely used in the Middle East and second-most in several other countries. The effect of WPS on normal development is not yet fully understood, thus the aim of this study is to explore the acute toxicity effects of WPS extract on zebrafish larvae. Methods: In this study, we compared the effects of WPS smoke condensates at concentrations varying from 50 to 200 µg/mL on developmental, cardiac, and behavioural (neurotoxicity) functions. Gene expression patterns of cardiac biomarkers were also evaluated by RT-qPCR. Results: Exposing zebrafish embryos to 50, 100, 150 and 200 µg/mL WPS for three days did not affect the normal morphology of Zebrafish embryos, as the tail flicking, behavioural and locomotion assays did not show any change. However, WPS deregulated cardiac markers including atrial natriuretic peptide (ANP/NPPA) and brain natriuretic peptide (BNP/NPPB). Furthermore, it induced apoptosis in a dose-dependent manner. Conclusion: Our data demonstrate that WPS can significantly affect specific cardiac parameters during the normal development of zebrafish. Further investigations are necessary to elucidate the pathogenic outcome of WPS on different aspects of human life, including pregnancy.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALSmodels and any C. elegans strain that exhibits altered motility.

Evaluation of Motor Impairment in <em>C. elegans</em> Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS models and any C. elegans strain that exhibits altered motility.

Lumbriculus variegatus: A novel organism for in vivo pharmacology education.

Pharmacology graduates require an understanding of both in vitro and in vivo drug responses but there has been a decline in animal use in pharmacology education over the last 30 years. To address this, we present the novel invertebrate model, Lumbriculus variegatus, for in vivo testing of drugs in a teaching environment. We have developed two novel behavioral assays: the stereotypical movement assay, which measures the effect of drugs on the ability of L. variegatus to perform stereotypical movements following tactile stimulation, and the free locomotion assay, which measures drug effects on unstimulated movement. We report the effects of compounds with diverse pharmacodynamic properties on L. variegatus using these assays. The ryanodine receptor antagonist, dantrolene, altered the unstimulated movement of L. variegatus at 5 μM, whereas stimulated movement was inhibited at ≥25 μM. Lidocaine, a voltage‐gated sodium channel blocker, and quinine, a nonselective sodium and potassium channel blocker, reduced both stimulated and unstimulated L. variegatus movement at ≥0.5 mM. Inhibitory effects of quinine persisted for up to 24 h after drug removal, whereas lidocaine effects were reduced 10 min after drug removal. Herein, we provide proof‐of‐concept utilization of L. variegatus as an organism for use in in vivo pharmacology education but without regulatory constraints or the need for specialized equipment and training.

Lipin 1 deficiency causes adult-onset myasthenia with motor neuron dysfunction in humans and neuromuscular junction defects in zebrafish.

Lipin 1 is an intracellular protein acting as a phosphatidic acid phosphohydrolase enzyme controlling lipid metabolism. Human recessive mutations in LPIN1 cause recurrent, early-onset myoglobinuria, a condition normally associated with muscle pain and weakness. Whether and how lipin 1 deficiency in humans leads to peripheral neuropathy is yet unclear. Herein, two novel compound heterozygous mutations in LPIN1 with neurological disorders, but no myoglobinuria were identified in an adult-onset syndromic myasthenia family. The present study sought to explore the pathogenic mechanism of LPIN1 in muscular and neural development.Methods: The clinical diagnosis of the proband was compared to the known 48 cases of LPIN1 recessive homozygous mutations. Whole-exome sequencing was carried out on the syndromic myasthenia family to identify the causative gene. The pathogenesis of lipin 1 deficiency during somitogenesis and neurogenesis was investigated using the zebrafish model. Whole-mount in situ hybridization, immunohistochemistry, birefringence analysis, touch-evoke escape response and locomotion assays were performed to observe in vivo the changes in muscles and neurons. The conservatism of the molecular pathways regulated by lipin 1 was evaluated in human primary glioblastoma and mouse myoblast cells by siRNA knockdown, drug treatment, qRT-PCR and Western blotting analysis.Results: The patient exhibited adult-onset myasthenia accompanied by muscle fiber atrophy and nerve demyelination without myoglobinuria. Two novel heterozygous mutations, c.2047A>C (p.I683L) and c.2201G>A (p.R734Q) in LPIN1, were identified in the family and predicted to alter the tertiary structure of LPIN1 protein. Lipin 1 deficiency in zebrafish embryos generated by lpin1 morpholino knockdown or human LPIN1 mutant mRNA injections reproduced the myotomes defects, a reduction both in primary motor neurons and secondary motor neurons projections, morphological changes of post-synaptic clusters of acetylcholine receptors, and myelination defects, which led to reduced touch-evoked response and abnormalities of swimming behaviors. Loss of lipin 1 function in zebrafish and mammalian cells also exhibited altered expression levels of muscle and neuron markers, as well as abnormally enhanced Notch signaling, which was partially rescued by the specific Notch pathway inhibitor DAPT.Conclusions: These findings pointed out that the compound heterozygous mutations in human LPIN1 caused adult-onset syndromic myasthenia with peripheral neuropathy. Moreover, zebrafish could be used to model the neuromuscular phenotypes due to the lipin 1 deficiency, where a novel pathological role of over-activated Notch signaling was discovered and further confirmed in mammalian cell lines.