Investigating Diseases of Dopaminergic Neurons and Melanocytes Using Zebrafish

Abstract

Zebrafish offer experimental advantages that have been exploited by developmental biologists and, ­increasingly, by those interested in disease mechanisms. Parkinson’s disease is characterized by the loss of dopaminergic neurons of the substantia nigra and motor symptoms such as slow movement, rigidity, and tremor. Traditionally investigators have used rodents and non-human primates as model animals to study the genetic and environmental causes of Parkinson’s diseases. However zebrafish offer an attractive alternative for behavioral studies because the larvae are small enough to fit into multi-well plates and thus are amenable to automated behavioral analysis. While there are significant anatomical differences between the zebrafish and human brains, in the zebrafish diencephalon there is a group of dopaminergic neurons whose projections are analogous to those of neurons in the human substantia nigra. Studies taking advantage of these features have measured the swimming behavior of zebrafish larvae in which expression of genes linked to familial Parkinson’s disease have been reduced, or that have been treated with pharmaceutical agents that target dopaminergic neurons. These studies have also included histological analyses of dopaminergic neurons. Zebrafish have also been used to dissect the genetic pathways that govern differentiation and survival of melanocytes. The assumption is that disruption of these pathways underlies diseases of melanocytes, which include vitiligo, a disease of melanocyte degeneration, and metastatic melanoma. It is believed that melanocytes and dopaminergic neurons must share vulnerability to particular mutations or environmental insults because risk for Parkinson’s disease and metastatic melanoma are associated with one another. Interestingly a mutagenesis screen in zebrafish may have identified one such shared requirement. In a forward screen, a mutant exhibiting melanocyte cell death was isolated and later shown to harbor a loss-of-function ­mutation in the gene encoding ion channel Transient Receptor Potential Melastatin-like 7 (TRPM7). TRPM7 was previously identified as possibly conferring risk for a Parkinsonian condition. This example reveals the potential for studies in zebrafish to reveal the genetic requirements of dopaminergic neurons, of melanocytes, and those shared by both cell types. This chapter provides the protocols used by our research group to examine the behavior of zebrafish larvae, and to monitor dopaminergic neurons and melanocytes by histology.