Burst Movements Research Articles

CRISPRa-Mediated Upregulation of scn1laa During Early Development Causes Epileptiform Activity and dCas9-Associated Toxicity.

Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene SCN1A. DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous SCN1A can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene scn1Laa in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9-VPR and dCas9-VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of scn1Laa mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published scn1Laa and scn1Lab loss-of-function models, we conclude that gain of scn1Laa function can have an equally severe phenotype. Upregulation of scn1Laa in the current zebrafish model for DS, scn1Lab-KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.

NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome.

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.

Biologging Tags Reveal Links Between Fine-Scale Horizontal and Vertical Movement Behaviors in Tiger Sharks (Galeocerdo cuvier)

An understanding of the role that large marine predators play in structuring trophic flow and nutrient cycling in marine ecosystems requires knowledge of their fine-scale (m-km) movement behaviours. In this study, biologging tags were used to reveal new insights into the three-dimensional fine-scale movement ecology of tiger sharks (Galeocerdo cuvier) at Ningaloo Reef, Western Australia. Tags deployed on 21 sharks in April-May 2017 for durations of 5-48 hours recorded both physical parameters such as depth and temperature, and, through the use of accelerometers, gyroscopes and compasses, in-situ measurements of animal trajectory and locomotion. Animal-borne-video enabled the validation of behavioural signatures, mapping of habitat, and recording of interactions with prey. Collectively, these data were used to examine the link between vertical (oscillations) and horizontal (tortuosity) movements, and link sensor data to prey interactions recorded by the video. This biologging approach revealed complex movements that would otherwise be invisible within the time-depth records provided by traditional tagging techniques. The rate of horizontal turning was not related to vertical oscillations, suggesting that vertical movements occur independently of searching behaviours in tiger sharks. These animals displayed tortuous movements possibly associated with prey searching for 27% of their tracks, and interactions with prey elicited varied responses including highly tortuous paths and burst movements. Accurate speed measurements and GPS anchor points will considerably enhance the value of magnetometer data in future studies by facilitating more accurate dead-reckoning and geo-referencing of area-restricted search behaviours.

A miniaturized threshold-triggered acceleration data-logger for recording burst movements of aquatic animals.

Although animal-borne accelerometers are effective tools for quantifying the kinematics of animal behaviors, quantifying the burst movements of small and agile aquatic animals remains challenging. To capture the details of burst movements, accelerometers need to sample at a very high frequency, which will inevitably shorten the recording duration or increase the device size. To overcome this problem, we developed a high-frequency acceleration data-logger that can be triggered by a manually defined acceleration threshold, thus allowing the selective measurement of burst movements. We conducted experiments under laboratory and field conditions to examine the performance of the logger. The laboratory experiment using red seabream (Pagrus major) showed that the new logger could measure the kinematics of their escape behaviors. The field experiment using free-swimming yellowtail kingfish (Seriola lalandi) showed that the loggers trigger correctly. We suggest that this new logger can be applied to measure the burst movements of various small and agile animals.

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays. Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction. In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Evaluation of spontaneous propulsive movement as a screening tool to detect rescue of Parkinsonism phenotypes in zebrafish models

Zebrafish models of human neuropsychiatric diseases offer opportunities to identify novel therapeutic targets and treatments through phenotype-based genetic or chemical modifier screens. In order to develop an assay to detect rescue of zebrafish models of Parkinsonism, we characterized spontaneous zebrafish larval motor behavior from 3 to 9 days post fertilization in a microtiter plate format suitable for screening, and clarified the role of dopaminergic signaling in its regulation. The proportion of time that larvae spent moving increased progressively between 3 and 9 dpf, whereas their active velocity decreased between 5 and 6 dpf as sporadic burst movements gave way to a more mature beat-and-glide pattern. Spontaneous movement varied between larvae and during the course of recordings as a result of intrinsic larval factors including genetic background. Variability decreased with age, such that small differences between groups of larvae exposed to different experimental conditions could be detected robustly by 6 to 7 dpf. Suppression of endogenous dopaminergic signaling by exposure to MPP +, haloperidol or chlorpromazine reduced mean velocity by decreasing the frequency with which spontaneous movements were initiated, but did not alter active velocity. The variability of mean velocity assays could be reduced by analyzing groups of larvae for each data point, yielding acceptable screening window coefficients; the sample size required in each group was determined by the magnitude of the motor phenotype in different models. For chlorpromazine exposure, samples of four larvae allowed robust separation of treated and untreated data points (Z = 0.42), whereas the milder impairment provoked by MPP + necessitated groups of eight larvae in order to provide a useful discovery assay (Z = 0.13). Quantification of spontaneous larval movement offers a simple method to determine functional integrity of motor systems, and may be a useful tool to isolate novel molecular modulators of Parkinsonism phenotypes.

Swimming behaviour of upriver migrating sea lamprey assessed by electromyogram telemetry

The main subject of this study was the swimming behaviour of upriver migrating sea lamprey, Petromyzon marinus, with particular focus on identification of their swim strategies to overcome areas of difficult passage. A biotelemetry technique (electromyogram telemetry) was used to register muscle activity of the tagged animals. In the 2005 spawning season, five adult sea lampreys were surgically tagged and released in the field. Before release, electromyogram (EMG) records were calibrated with the P. marinus swimming speed in a swim tunnel. Differences between ground speed and swimming speed in the wild suggest that the calibrated CEMG (coded electromyogram) transmitter output corresponds to an activity index, and cannot be properly related to actual swimming speed. This study notes the need to confirm the laboratory calibration curves, to ascertain their use in determining swimming speed of tagged fish in the wild. In 2006, in order to confirm the field results seven adult sea lampreys were tagged, calibrated in the laboratory and released in a 30-m long experimental outdoor canal. The results were similar: observed swimming speed was generally higher when compared with the swimming speed obtained with the EMG signal. In the river, when swimming through slow-flow stretches, sea lampreys maintained a constant pattern of activity, attaining an average ground speed of 0.76 BL s−1 (2.5 km h−1). When sea lampreys encountered rapid flow reaches they alternated between short movements (c. 67 s) and periods of rest (c. 99 s). In each swim bout they progressed approximately 14 m; to overcome more difficult obstacles sea lampreys increased their number of burst movements instead of longer or more violent swimming events. About 43% of the time negotiating difficult passage areas was spent in resting by attaching motionless to the substrate with their oral disk.

Longer flumes increase critical swimming speeds by increasing burst–glide swimming duration in carp Cyprinus carpio, L.

Carp Cyprinus carpio altered the repertoire of swimming behaviour with increased flume length. While the transition speed from steady to burst–coast swimming was unaffected by flume length, fish reached higher critical swimming speed (Ucrit), consequently swimming for longer periods of time in burst–coast mode and hence performing more work before becoming fatigued. Analysis of swimming behaviour of burst–coast swimming revealed an increase in duration and a decrease in distance of forward burst movements with increasing water speeds. Frequency was unaffected by water speed. Overall, longer flumes increased Ucrit by allowing for less restricted burst–coast swimming behaviour.

The long and winding road: influences of intracellular metabolite diffusion on cellular organization and metabolism in skeletal muscle

A fundamental principle of physiology is that cells are small in order to minimize diffusion distances for O(2) and intracellular metabolites. In skeletal muscle, it has long been recognized that aerobic fibers that are used for steady state locomotion tend to be smaller than anaerobic fibers that are used for burst movements. This tendency reflects the interaction between diffusion distances and aerobic ATP turnover rates, since maximal intracellular diffusion distances are ultimately limited by fiber size. The effect of diffusion distance on O(2) flux in muscle has been the subject of quantitative analyses for a century, but the influence of ATP diffusion from mitochondria to cellular ATPases on aerobic metabolism has received much less attention. The application of reaction-diffusion mathematical models to experimental measurements of aerobic metabolic processes has revealed that the extreme diffusion distances between mitochondria found in some muscle fibers do not necessarily limit the rates of aerobic processes per se, as long as the metabolic process is sufficiently slow. However, skeletal muscle fibers from a variety of animals appear to have intracellular diffusion distances and/or fiber sizes that put them on the brink of diffusion limitation. Thus, intracellular metabolite diffusion likely influences the evolution of muscle design and places limits on muscle function.

Behavioural patterns of sea lampreys’ spawning migration through difficult passage areas, studied by electromyogram telemetry

Electromyogram telemetry (EMG) was used to study the behaviour of sea lamprey Petromyzon marinus during migration through difficult passage areas. When swimming on stretches free from obstacles, the EMG values of sea lampreys showed a constant pattern of activity. Conversely, when negotiating difficult passage areas their behaviour were characterized by alternating between short bursts of intense activity (i.e. burst movements) and periods of motionless rest during which sea lampreys attached to convenient structures by means of the oral sucker. During blockstone weirs negotiation, tagged sea lampreys spent on average 23% of the time swimming rapidly by means of multiple bouts of burst movements of 31 s duration. To recover from each burst movement, the sea lampreys spent on average 2 min 25 s resting. Sea lampreys seemed affected by increasing fatigue, which probably resulted from initiating a new burst movement without fully recovering physiologically from the previous efforts.