Normalization of large-scale behavioural data collected from zebrafish

Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour

Population relevance of neurotoxic effects in refined and alternative behavior tests with zebrafish (Danio rerio)

Water supplies are widely, but unobtrusively contaminated with numerous substances of largely unknown biological properties. A particularly worrisome group are neurotoxic substances, which may, in the long term, not only affect human health, but also wildlife. Neurotoxic effects have become an issue of emerging concern in ecotoxicology, since they may have multiple underlying mechanisms, are often hard to detect, but have the potential to give rise to a severe adverse outcome. As neurotoxicity is even more difficult to detect without extensive animal testing, it presents a major challenge to modern ecotoxicology which is striving to reduce and replace animal studies. My model species, the zebrafish (Danio rerio), is widely used in aquatic ecotoxicology but room for refinement remains especially where tests are carried out with adult individuals instead of potentially less perceptive early-life stages. Since zebrafish, like many other small fish, naturally form shoals and likely behave differently in isolation, I developed a shoal-based approach. In brief, early-life stage tests according to OECD TG 210 were augmented by two behavior tests that are typically carried out with single adult fish, but could be adapted to groups of juveniles with acceptable limitations: a novel tank test and a predator response assay. The selective serotonin reuptake inhibitor fluoxetine ((RS)-N-Methyl-3-phenyl-3-(4-trifluoromethylphenoxy) propylamine) served as model substance during a proof-of-concept study. In a follow-up study, I verified the suitability of this approach using a selection of other substances with different modes-of-action: carbamazepine (sodium channel inhibition), paraoxon-methyl (acetylcholine esterase inhibition), and tris(1,3-dichlorisopropyl) phosphate (TDCPP; endocrine disruption). Finally, in order to assess whether existing alternative methods correlate to immediately population relevant endpoints, I carried out several other experiments across the life-stages of zebrafish with the same model substances. Fluoxetine produced adverse effects down to concentrations three orders of magnitude below the EC10 from acute fish embryo toxicity tests (OECD TG 236). The known neurotoxicants carbamazepine and paraoxon-methyl caused significant effects on zebrafish behavior both upon release into a novel tank and after presentation of a predator dummy. TDCPP, which is thought to disrupt neural development at much earlier stages than those exposed in my experiments, only caused minor behavioral changes. Histopathology of the test fish confirmed the absence of acute organ damage at the concentrations used (always ≤ EC10 from fish embryo tests). The suitability of shoal-based behavioral changes in juvenile zebrafish as sensitive endpoints of neurotoxicity could thus be confirmed. The deviations in behavior compared to the control groups permit conclusions about the “anxiety state”, which arguably influences the fish’s survival chances in the wild. An early and more abstract behavior endpoint, larval motility (6 dpf), also proved to be very efficient and held up well in a comparison with adult and juvenile behavior tests. Finally, a reproduction assay with adult fish exposed to fluoxetine revealed decreased fecundity as another directly population relevant effect of this chemical. 2 Correlation with embryonic and further adult data from literature revealed the good predictive power of 24-h spontaneous coiling tests for later behavior defects, leading me to propose a set of embryonic tests (FET + coiling) for neurotoxicity range-finding and screening in the future. If the results from these “alternative methods” are negative or inconclusive, in vivo testing is indispensable to assess neurotoxicity; as such, larval motility and juvenile behavior assays might follow

Zebrafish Swim Capacity, Gait, and Posture During Innate Spinal Cord Repair

Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Significant advances for understanding the cellular and molecular mechanisms of innate spinal cord repair are best supported by accurate assessment of swim function. This study introduces neuromuscular-associated measurements derived from gait and posture, compares new and classical functional measurements longitudinally, and provides a comprehensive correlative analysis of various functional and cellular regeneration outputs. It also explores trajectories of functional regeneration through tensor decomposition and demonstrates that swim function at 2 weeks post-injury can be used to predict 8-week regeneration outcomes. By establishing swim quality metrics which are both distinct from previous measurements and demonstrably more associated with glial bridging and axon regrowth, this study has the potential to accelerate discovery and reduce redundancy in future studies. Software for this study, featuring zebrafish swim quality analysis, is now freely available

Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep.

Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriv- ing from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation...

Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients