Transgenic Zebrafish Expressing mCherry in the Mitochondria of Dopaminergic Neurons

Zebrafish. 2015 Oct;12(5):349-56. doi: 10.1089/zeb.2015.1085. Epub 2015 Sep 10.

Abstract

Genetic mutations and environmental toxins are known to affect mitochondrial health and have been implicated in the progressive degeneration of dopaminergic neurons in Parkinson's disease. To visualize mitochondria in dopaminergic neurons of live zebrafish, we used the regulatory elements of the dopamine transporter (dat) gene to target a reporter, mCherry, after fusion with the mitochondrial localizing signal (MLS) of Tom20. Immunoblot analysis of mitochondrial and cytosolic fractions from Tg(dat:tom20 MLS-mCherry) larvae shows that mCherry is efficiently targeted to the mitochondria. Confocal imaging of live fish was carried out from 1 day postfertilization (dpf) to 9 dpf. We also colocalized dat mRNA expression with the mCherry protein in the olfactory bulb (OB), subpallium (SP), pretectum (Pr), diencephalic clusters 2 and 3 (DC2/3), caudal hypothalamus (Hc), locus coeruleus (LC), anterior preoptic area (POa), retinal amacrine cells (RAC), caudal hypothalamus (Hc), and preoptic area (PO). Treating Tg(dat:tom20 MLS-mCherry) larvae with the dopaminergic neurotoxin MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) at 2 or 3 dpf resulted in a decrease in mCherry fluorescence in the pretectum, olfactory bulb, subpallium, diencephalic clusters 2 and 3, and the caudal hypothalamus. Labeling of mitochondria in nigrostriatal dopaminergic neurons of zebrafish could allow their visualization in vivo following genetic or pharmacological manipulations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / pharmacology*
  • Animals
  • Animals, Genetically Modified / genetics
  • Animals, Genetically Modified / metabolism
  • Dopamine / metabolism
  • Dopamine Plasma Membrane Transport Proteins / genetics*
  • Dopamine Plasma Membrane Transport Proteins / metabolism
  • Dopaminergic Neurons / metabolism*
  • Fluorescence
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mitochondria / drug effects*
  • Mitochondria / metabolism
  • Neurotoxins / pharmacology*
  • Organ Specificity
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Zebrafish / genetics*
  • Zebrafish / metabolism
  • Zebrafish Proteins / genetics*
  • Zebrafish Proteins / metabolism

Substances

  • Dopamine Plasma Membrane Transport Proteins
  • Luminescent Proteins
  • Neurotoxins
  • Receptors, Cytoplasmic and Nuclear
  • SLC6A3 protein, zebrafish
  • Zebrafish Proteins
  • red fluorescent protein
  • tomm20b protein, zebrafish
  • 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • Dopamine