Characterization of genetically labeled dopamine neurons and circadian studies of the zebrafish retina

Abstract

Dopamine plays key roles in many basic functions in the central nervous system. In order to study developmental and functional roles of dopaminergic cells in the zebrafish, we have examined a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (TH) promoter. TH-driven GFP was expressed in cells located in the inner nuclear layer. Immunocytochemistry with antibodies for GFP and TH showed that 29 ± 2% of GFP-labeled cells also expressed TH. Loose-patch voltage-clamp recording from GFPlabeled cells revealed that these dopaminergic neurons are spontaneously active in darkness. This transgenic line provides a useful tool to target retinal dopaminergic cells in vivo and in situ.

The vertebrate retina is profoundly influenced by circadian rhythmicity, yet little is known about the mechanisms of the zebrafish retinal circadian clock. To further the study of the zebrafish retinal clock, we have constructed a recombinant BAC in which short-half life GFP is under the control of the zebrafish circadian gene Per3 promoter. Expression of the modified BAC clone was observed in injected zebrafish embryos. Further intercrossing of injected fish and screening of their progeny may identify transgenic Per3::d2GFP fish. Meanwhile, by using transgenic Per3::LUC fish (generated by G. Cahill, University of Houston), we are able to examine the effect of light stimuli on bioluminescence rhythm at different times of a circadian cycle. Our results demonstrate that cultured zebrafish retina shows large phase shifts with phaseresponse curve close to type 0.