Using Zebrafish for Screening and Development of New Nicotinic and Dopaminergic Drugs

Abstract

Zebrafish are a new vertebrate animal model with advantages for screening and development of therapeutic agents. Many biochemical pathways present in humans are conserved in zebrafish. Zebrafish embryos develop rapidly (first somite at about 10 hours of development compared to 9-10 days in the rat) outside of the mother with the potential for generating hundreds of embryos from a single mating. Zebrafish can be grown in plates for several days to allow easy observation and use in High-Throughput Screening (HTS). The early embryos are also transparent, providing the ability to observe specific cells or brain regions during development without the need to kill the animals. Embryogenesis is complete by 72 hours post fertilization (hpf) and most organs are fully developed at 96 hpf [1]. A large number of mutants are available and much of the genome has been sequenced (http://www.sanger.ac.uk) which allows the genetic advantages of mice, but with a short generation time (2-3 months). Zebrafish neural development occurs in a well characterized pattern and well-defined molecular markers (antibodies, DNA probes) are available to help identify specific cells and brain regions. Transgenic zebrafish have been developed which express GFP in specific cells [2] and allow for the activity of specific neurons to be monitored in vivo [3]. The small size of zebrafish makes them ideal for use in 96 or 384 well plates. The cost to maintain zebrafish is a fraction of that for mice since they don’t require feeding until 6-7 days post fertilization. The use of zebrafish allows one to combine the ability to perform behavioral assays with HTS [4]. Many behavioral assays developed in other animals, and which are used to assay drugs targeted to several neurological diseases, are available in zebrafish [5]. These include Prepulse Inhibition (PPI), startle response, locomoter and learning assays. These can be performed on a higher scale and at much lower cost than in other vertebrates. The data collection and analysis from many of these tests can be automated [4]. Indeed, zebrafish can be used at several points in the drug discovery process. Zebrafish are ideal for testing drug toxicity on a large scale, thus saving much time, money and effort to further develop a compound with toxicity in vertebrates. In many cases, compounds with known toxicity in humans have a similar response in zebrafish [1]. Zebrafish have also been used in target confirmation after a lead compound has been identified. In vivo Structure Activity Relationship (SAR) studies in zebrafish have been performed [6]. Using in vivo screens during early stages of drug discovery could produce great savings in time, money and efficiency. Zebrafish have been used already in a large number of small molecule screens in both wild-type and mutant fish [1]. Zebrafish have also been used for targeted screens as well. Methodologies are being developed for rapid behavior-based identification of neuroactive small molecules in zebrafish [7]. Thus it may be useful to screen and develop drugs to treat illnesses related to cholinergic and dopaminergic systems in zebrafish. Neuronal Nicotinic Acetylcholine Receptors (nAChRs) are involved in several CNS disorders such as addiction, Alzheimer’s, epilepsy, and schizophrenia period [8].