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Abstract:
Purpose: Dopamine plays key roles in a variety of basic functions in the central nervous system. To study developmental and functional roles of dopaminergic cells in zebrafish, we have generated a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (th1) promoter.
Methods: A 12 kb gene fragment that contains the th1 promoter was isolated and ligated to the MmGFP coding sequence, linearized, microinjected into 1−2 cell stage embryos and the founders crossed with wild−type fish to screen for transgenic lines. Tg(−12th:MmGFP) embryos were visualized under fluorescence microscopy for GFP expression during development. Confocal microscopy was used to visualize GFP−labeled cells in the living whole mount retina and immunostained vertical sections of adult zebrafish retina. Single−cell reverse transcription polymerase chain reaction (RT−PCR) was performed on individual GFP+ cells collected from dispersed retinal cell cultures for th1 and dopamine transporter (dat). Loose−patch recordings of spike activity of GFP+ neurons were made in isolated whole mount retinas.
Results: th1 promoter−driven GFP exhibited robust expression in the brain and retina during zebrafish development. In juvenile and adult fish retinas, 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. Two subpopulations of GFP−labeled cells were identified by fluorescent microscopy: bright GFP−expressing cells and dim GFP−expressing cells. Seminested single−cell RT−PCR showed that 71% of dim GFP−expressing cells expressed both th and dat mRNA. Loose−patch voltage−clamp recording from dim GFP−labeled cells in retinal whole mounts revealed that many of these dopaminergic neurons are spontaneously active in darkness.
Conclusions: Although this Tg(−12th:MmGFP) line is not a completely specific reporter for dopaminergic neurons, using relative GFP intensity we are able to enrich for the selection of retinal dopaminergic cells in vitro and in situ in molecular and electrophysiological experiments. This transgenic line provides a useful tool for studying retinal dopaminergic cells in the zebrafish
