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

Abstract

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.

Introduction

The long-term aim of our work is to develop improved treatments for neurological diseases associated with movement disorders. The zebrafish presents some methodological advantages as a tool to achieve this goal, including the unique applicability of high-throughput screening in a vertebrate model in vivo. Zebrafish larvae can be housed in 96-well plates, allowing libraries of small molecules to be assayed against phenotypes in order to identify compounds with disease-modifying potential (Zon and Peterson, 2005). Importantly, this can be carried out without making prior assumptions regarding drug targets, which might otherwise limit opportunities for discovery. Consequently, there is much interest in exploiting zebrafish models for the analysis of diseases and possible identification of new treatments. Phylogenetic conservation of many molecular, cellular and neuroanatomical features suggests that the zebrafish CNS presents a suitable biochemical and tissue environment to model human neurological disease, a prediction supported by recent studies showing that genetic or chemical manipulation of zebrafish can recapitulate key features of relevant human disorders (Bandmann and Burton, 2010, Panula et al., 2010). Full exploitation of zebrafish neurological disease models for discovery of small molecule and genetic modifiers will be critically dependent on selection of appropriate phenotypic assays. In vivo models of neurological diseases have inherent advantages for modeling numerous aspects of disease pathogenesis, and also provide opportunities to use functional and behavioral assays as endpoints. The latter will be of particular importance in the study of diseases whose clinical manifestations are caused by disturbances in the physiology of neural circuits rather than neuronal cell loss, for example primary dystonia (Tanabe et al., 2009). Morphological end points might be uninformative for the analysis of these disorders, whereas physiological assays may be helpful.

Several different types of motor behavior can be quantified in zebrafish larvae, including spontaneous movement and evoked responses. The latter range in complexity, from reflex reactions to acoustic or cutaneous stimuli, to neurobehavioral traits relating to visual assessment of novel environmental cues or social behavior (Orger et al., 2004). Genetic (Muto et al., 2005, Peng et al., 2009) and chemical (Kokel et al., 2010, Rihel et al., 2010) screens relying on neurological or behavioral assays have recently been reported, demonstrating the feasibility of screening using functional end points. It is possible that a parallel approach might be used in genetic and chemical modifier screens to detect rescue of neurological phenotypes in disease models, raising the exciting prospect of phenotype-driven identification of novel therapeutic leads and drug targets in vivo. For the development of methodology to exploit zebrafish models of movement disorders for screening, we reasoned that an assay should be automated and applicable at larval time points at which high-throughput screening would be feasible, and at which pathogenesis might occur. In addition, the assay should quantify functions relevant to neural circuits implicated in pathogenesis, including the dopaminergic system that is centrally involved in many human movement disorders, and should yield quantitative data that could reliably detect a realistic level of partial phenotypic rescue.

After zebrafish embryos hatch, larval tail beating results in spontaneous propulsive movement (Drapeau et al., 2002). Previous work has shown that larval movement is modulated by dopaminergic function. The neurotoxins MPTP and MPP⁺ reduced both the number of dopamine neurons (Bretaud et al., 2004, Lam et al., 2005, McKinley et al., 2005, Sallinen et al., 2008) and the total dopamine content (Sallinen et al., 2008) of larval zebrafish; exposure to these agents was associated with reduced motor activity, measured as the distance moved spontaneously over the course of an assay at 7 dpf (Sallinen et al., 2008), or as abnormalities of trunk movements in response to cutaneous stimuli in immobilized samples at 72 hpf (Lam et al., 2005). Similarly, dopamine D2 receptor antagonist drugs reduced displacement of 7 dpf and 14 dpf larvae over a 5-minute recording (Giacomini et al., 2006). These studies did not differentiate a hypokinetic phenotype (reduced number of movements) from a bradykinetic phenotype (executed movements are slower) in animals with impaired dopaminergic function. Furthermore, there is little published information on how spontaneous zebrafish motor behavior develops quantitatively during later larval stages, when neurodegenerative phenotypes realistically might become apparent, and little is known about how genetic background and environmental factors affect spontaneous movement and its variability. This information will be critical to understanding how spontaneous movement might be used as an assay for screening applications in Parkinsonism models. The purpose of this study was to quantify the development of spontaneous propulsive movement in zebrafish, to understand its sources of variability and the role of dopaminergic neurotransmission in its regulation, and to evaluate the suitability of spontaneous movement assays to detect rescue of motor function in a screening paradigm.