ZEBRAFISH AS A MODEL SYSTEM TO STUDY DOPAMINE-RELATED NEUROLOGICAL DISORDERS
Open Access
- Author:
- Boehmler, Wendy Ann
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 21, 2006
- Committee Members:
- Robert G Levenson, Committee Chair/Co-Chair
Victor Alan Canfield, Committee Member
Keith C Cheng, Committee Member
John Ellis, Committee Member - Keywords:
- zebrafish
dopamine receptor
mRNA expression
behavior
adenosine receptor - Abstract:
- The mechanism by which brain circuitry is wired to produce various functional outputs such as emotion, locomotion, learning and memory remains poorly understood. Dysregulation of these processes can lead to neuropsychiatric and neurodegenerative disorders such as schizophrenia and Parkinson’s disease. While both of these diseases are related to dysfunction in dopamine signaling, genetic analyses have shown no alterations in the genes encoding dopamine receptors in patients with these diseases. In addition, the mechanisms underlying aberrant dopaminergic signaling in these neuropyschiatric disorders remain enigmatic. Available treatments relieve the primary symptoms of these neurological disorders, but introduce a new host of unwanted side effects. Therefore, it is important to identify new targets new treatments for these patients. In order to determine the molecular mechanisms involved in these neuropathogies, it will be crucial to have a valuable animal model system. Zebrafish have emerged as an important vertebrate model system not only to identify new genes and determine their function, but also as a high throughput model to identify new drugs and their targets. The main goal of our research is to identify and characterize the D2-like dopamine receptors in zebrafish. Furthermore, we hypothesize that the identification and characterization of the molecular components of the DA system in zebrafish will lead to new insights regarding regulation of dopamine signaling. The zebrafish model system provides several advantages for identifying and characterizing genes and their function. The zebrafish embryo develops ex utero and is transparent, which allows for gene expression analysis using whole-mount in situ hybridization. Zebrafish are amenable to rapid forward and reverse genetic techniques. Mutant zebrafish can be screened morphologically and behaviorally to identify new genes and their function. Antisense morpholino oligonucleotides make it possible to knock down expression of any specified gene during the first several days of development, in order to determine its function. Zebrafish display a cohort of behaviors related to DA signaling, including locomotion and conditioned place preference. Additionally, chemicals and drugs can be added directly to the water, which makes zebrafish an attractive model system for toxicology and drug screening. This work has focused on two families of G-protein-coupled receptors; the D2-like dopamine receptors and the A2-like adenosine receptors. Both families of receptors have been implicated in dopamine-related neurological disease. To better understand the function of these genes, we have cloned and characterized these genes in zebrafish. In order to identify dopamine receptor genes, we performed searches of the zebrafish genomic sequence database, which yielded contigs containing segments of several D2-like dopamine receptor genes. From these sequences, we amplified full-length cDNAs encoding three D2, one D3, and three D4 receptor subtypes via RT-PCR. The predicted proteins ranged from 57-72% amino acid identity when compared to the human dopamine receptors. Zebrafish dopamine receptor genes were mapped by using the T51 radiation hybrid panel. With the exception of drd2b and drd4b, the remaining dopamine receptor genes mapped to positions found to be syntenic to regions of human chromosomes containing the orthologs of these dopamine receptor genes. To further characterize these genes, whole-mount in situ hybridization was used to investigate their expression during development. All of the receptor genes were found predominantly throughout the central nervous system and exhibit distinct but overlapping expression patterns during embryogenesis. In order to analyze the function of the dopamine receptors identified, we examined the effect of the atypical antipsychotic clozapine (a D4R antagonist) on swimming behavior of zebrafish larvae. Clozapine produced a rapid and profound sedative-like effect on the fish. This response was dose-dependent and reversed by ABT 724 (a selective D4R agonist), but was not reversed by quinpirole (a D2/D3R agonist). The response of zebrafish to clozapine provides a novel platform for performing genetic screens designed to identify genetic components involved in D4-mediated dopaminergic signaling. In the final chapter, we explore the mechanism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is commonly used to model Parkinson’s disease in various laboratory animals. Zebrafish are susceptible to MPTP, which induces the selective loss of dopaminergic neurons. Caffeine, an A1/A2a adenosine receptor antagonist, has neuroprotective properties against the toxicity of MPTP in mammals. In order to determine whether caffeine is neuroprotective in zebrafish, embryos were co-incubated with MPTP and caffeine. The presence of the dopamine transporter gene, which specifically labels dopaminergic neurons, suggests a neuroprotective effect of caffeine against MPTP in zebrafish embryos. This assay suggests zebrafish can be used to screen drugs with potential neuroprotective activity. Neuroprotection of caffeine is believed to be mediated through the direct antagonism of the A2a adenosine receptors. In order to determine whether these receptors exist in zebrafish, we performed searches of the zebrafish genome database which lead to the identification and cloning of two A2a and one A2b AR genes. These studies suggest that a similar mechanism of dopaminergic neuroprotection may exist in zebrafish.