1st INCF Workshop on Genetic Animal Models for Brain Diseases

The INCF Secretariat organized a workshop to focus on the “role of neuroinformatics in the processes of building, evaluating, and using genetic animal models for brain diseases” in Stockholm, December 13–14, 2009. Eight scientists specialized in the fields of neuroinformatics, database, ontologies, and brain disease participated together with two representatives of the National Institutes of Health and the European Union, as well as three observers of the national INCF nodes of Norway, Poland, and the United Kingdom

Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior

A Novel Long-term, Multi-Channel and Non-invasive Electrophysiology Platform for Zebrafish.

Zebrafish are a popular vertebrate model for human neurological disorders and drug discovery. Although fecundity, breeding convenience, genetic homology and optical transparency have been key advantages, laborious and invasive procedures are required for electrophysiological studies. Using an electrode-integrated microfluidic system, here we demonstrate a novel multichannel electrophysiology unit to record multiple zebrafish. This platform allows spontaneous alignment of zebrafish and maintains, over days, close contact between head and multiple surface electrodes, enabling non-invasive long-term electroencephalographic recording. First, we demonstrate that electrographic seizure events, induced by pentylenetetrazole, can be reliably distinguished from eye or tail movement artifacts, and quantifiably identified with our unique algorithm. Second, we show long-term monitoring during epileptogenic progression in a scn1lab mutant recapitulating human Dravet syndrome. Third, we provide an example of cross-over pharmacology antiepileptic drug testing. Such promising features of this integrated microfluidic platform will greatly facilitate high-throughput drug screening and electrophysiological characterization of epileptic zebrafish

Quantitative cross-species extrapolation between humans and fish: The case of the anti-depressant fluoxetine

This article has been made available through the Brunel Open Access Publishing Fund.Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 μg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation

Reduction of the ATPase inhibitory factor 1 (IF1) leads to visual impairment in vertebrates

In vertebrates, mitochondria are tightly preserved energy producing organelles, which sustain nervous system development and function. The understanding of proteins that regulate their homoeostasis in complex animals is therefore critical and doing so via means of systemic analysis pivotal to inform pathophysiological conditions associated with mitochondrial deficiency. With the goal to decipher the role of the ATPase inhibitory factor 1 (IF1) in brain development, we employed the zebrafish as elected model reporting that the Atpif1a−/− zebrafish mutant, pinotage (pnttq209), which lacks one of the two IF1 paralogous, exhibits visual impairment alongside increased apoptotic bodies and neuroinflammation in both brain and retina. This associates with increased processing of the dynamin-like GTPase optic atrophy 1 (OPA1), whose ablation is a direct cause of inherited optic atrophy. Defects in vision associated with the processing of OPA1 are specular in Atpif1−/− mice thus confirming a regulatory axis, which interlinks IF1 and OPA1 in the definition of mitochondrial fitness and specialised brain functions. This study unveils a functional relay between IF1 and OPA1 in central nervous system besides representing an example of how the zebrafish model could be harnessed to infer the activity of mitochondrial proteins during development

Zebrafish Models in NeuroPsychopharmacology and CNS Drug Discovery

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets

Population-scale organization of cerebellar granule neuron signaling during a visuomotor behavior.

Granule cells at the input layer of the cerebellum comprise over half the neurons in the human brain and are thought to be critical for learning. However, little is known about granule neuron signaling at the population scale during behavior. We used calcium imaging in awake zebrafish during optokinetic behavior to record transgenically identified granule neurons throughout a cerebellar population. A significant fraction of the population was responsive at any given time. In contrast to core precerebellar populations, granule neuron responses were relatively heterogeneous, with variation in the degree of rectification and the balance of positive versus negative changes in activity. Functional correlations were strongest for nearby cells, with weak spatial gradients in the degree of rectification and the average sign of response. These data open a new window upon cerebellar function and suggest granule layer signals represent elementary building blocks under-represented in core sensorimotor pathways, thereby enabling the construction of novel patterns of activity for learning

17α-ethinyl estradiol induces behavioral and reproductive damages in zebrafish (Danio rerio)

Synthetic estrogen 17α-ethinyl estradiol (EE2) is recognized for its ability to cause endocrine disruption in aquatic organisms and its high resistance to environmental degradation, being found in Brazilian waterbodies up to 100 ng L-1. Thus, the aim of this study was to evaluate the exploratory behavior and the histological effects on zebrafish gonads, caused by environmentally relevant concentrations of EE2. Females were exposed to nominal concentrations of 25 ng L-1 and 100 ng L-1 EE2 for 21 days. After that, a behavioral and histological analysis of gonads was performed. The EE2 caused the reduction of the exploratory activity of the animals, when observed the behavior, and an acceleration of the maturation process of the oocytes evaluated in the histological analysis. Considering the results obtained after the exposure to EE2, we highlight the concern about the potential risks it may cause to the aquatic biota, alerting to the need to control and monitor these micropollutants present in Brazilian water resources

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