Effects of 6-hydroxydopamine on primary cultures of substantia nigra: specific damage to dopamine neurons and the impact of glial cell line-derived neurotrophic factor.
6-Hydroxydopamine (6-OHDA)-induced loss of dopamine (DA) neurons has served to produce an animal model of DA neuron loss in Parkinson's disease. We report here the use of 6-OHDA to produce an in vitro model of this phenomena using dissociated cultures prepared from neonatal rat mesencephalon. Cultures were exposed to 6-OHDA (40-100 microm, 15 min) in an antioxidant medium, and DA and GABA neurons evaluated by immunocytochemistry. 6-OHDA induced morphological and biochemical signs of cell death in DA neurons within 3 h, followed by loss of tyrosine hydroxylase immunoreactive neurons within 2 days. In substantia nigra (SN) cultures, DA neurons were much more affected by 6-OHDA than were GABA neurons. In contrast, DA neurons from the ventral tegmental area were only lost at higher, non-specific concentrations of 6-OHDA. The effects of 6-OHDA on nigral DA neurons were blocked by inhibitors of high affinity DA transport and by z-DEVD-fmk (150 microm), a caspase inhibitor. Glial cell line-derived neurotrophic factor (GDNF) treatment reduced TUNEL labeling 3 h after 6-OHDA exposure, but did not prevent loss of DA neurons at 48 h. Thus, 6-OHDA can selectively destroy DA neurons in post-natal cultures of SN, acting at least in part by initiating caspase-dependent apoptosis, and this effect can be attenuated early but not late by GDNF.
Ding
Yun Min
YM
Department of Neurology and the Pittsburgh Institute for Neurodegenerative Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Jaumotte
Juliann D
JD
Signore
Armando P
AP
Zigmond
Michael J
MJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors; D008954:Models, Biological; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D016627:Oxidopamine / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013378:Substantia Nigra / Q000166:cytology / Q000187:drug effects*; D017557:Ventral Tegmental Area / Q000166:cytology / Q000187:drug effects
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D004791:Enzyme Inhibitors; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D016627:Oxidopamine