Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

Abstract

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

“…Zebrafish embryos were bathed in 1 μM benomyl or vehicle (0.01% DMSO) in E3 medium for zebrafish embryos from 5 h until 120 h postfertilization, anesthetized using tricaine methanesulfonate (0.02%), fixed in paraformaldehyde (4%) overnight, and mounted in agarose (1.2% wt/vol) for confocal imaging at 20× magnification. Zebrafish expressing GFP tagged to vesicular monoamine transporter protein (ETvmat2:GFP) were used to identify VMAT2 + (dopaminergic, (nor)adrenergic, serotonergic) neurons in whole embryos (26). Approximately 100 optical sections were gathered for each embryo, spaced 1.34 μM apart, using a Zeiss LSM 5 Pascal inverted microscope.…”

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

“…ddc is expressed in the th2-positive areas in adult (27)(28)(29)37), but its expression in embryos is restricted to the caudal tuberculum dopaminergic neuron and hindbrain raphe nucleus (14), where th2 is not expressed. In contrast, vmat2 is expressed in the diencephalon with a similar pattern of both th2 and 5-HT (17,26,30,31). We therefore selected vmat2 as the marker gene to determine co-localization of th2 and 5-HT.…”

Zebrafish as a Model for Developmental Neurotoxicity Assessment: The Application of the Zebrafish in Defining the Effects of Arsenic, Methylmercury, or Lead on Early Neurodevelopment

“…It seems clear that MPTP induces neurobehavioral changes and/or neuronal decrease in the developing zebrafish [37,43,62]. In Bretaud et al [37], while paraquat or rotenone treatment did not induce significant changes, MPTP exposure induced decreases in swimming speed and in the number of dopaminergic neurons in the diencephalon of zebrafish at 168 hpf and 120 hpf, respectively.…”

“…The zebrafish model has been applied for testing the developmental neurotoxic effects of exposure to chemicals related to Parkinson's disease, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), paraquat, and rotenone [37,62]. It seems clear that MPTP induces neurobehavioral changes and/or neuronal decrease in the developing zebrafish [37,43,62].…”