Abstract
Purpose: :
Dopamine is an important neurotransmitter in the central and peripheral nervous system. In the vertebrate retina, dopamine regulates many processes such as signal transmission and light adapting. In order to identify dopaminergic cells in vivo, transgenic zebrafish, in which green fluorescence protein (GFP) is driven by the promoter for tyrosine hydroxylase (TH), were generated by the Driever lab. We have examined the location and morphology of GFP labeled cells, as well as determined the proportion of cells exhibiting both TH and GFP immunoreactivity.
Methods: :
Heterozygous TH::GFP transgenic zebrafish were crossed with wild type AB* fish. Eggs were treated with 0.003% phenyl–2–thiourea and screened by visualizing the GFP expression in the embryonic brain at 30 hpf under fluorescence microscopy. Confocal microscopy was used to visualize GFP–labeled cells in the living wholemount retina and immunostained vertical sections of adult zebrafish retina. For immunocytochemistry, eyes of one–month old GFP–positive fish were removed and the eyecups were cryosectioned at 15 µm. Cryostat sections were then double stained with antibodies for GFP and TH. Immunohistochemistry on wholemount retina was performed similarly.
Results: :
TH–driven GFP had a robust expression in the brain and retina at 30hpf. In the 1–month–old zebrafish retina, GFP was expressed in cells located mainly in the inner nuclear layer (INL), with dense processes ramifying in the inner plexiform layer. GFP labeled cells have a density of 299 ± 48 cells/mm2 throughout the retina. The cell size was relatively even with a diameter ∼8 µm. Immunocytochemistry with antibodies for GFP and TH showed that 47.9 ± 6.7% of GFP labeled cells also express TH.
Conclusions: :
Our results indicate that the TH::GFP labeled cells are located in INL, which is consistent with their putative identification as interplexiform cells. Half of the cells co–express TH–driven GFP and TH protein, suggesting that the chance of identifying dopaminergic neurons is increased in vitro and in vivo. This transgenic fish line may be further used for developmental and functional studies of dopaminergic cells in zebrafish retina and brain.
Keywords: dopamine • retina