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 Yun Min Ding, 1 Juliann D. Jaumotte, Armando P. Signore and Michael J. Zigmond Department of Neurology and the Pittsburgh Institute for Neurodegenerative Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Abstract 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 mesenceph- alon. Cultures were exposed to 6-OHDA (40–100 lM, 15 min) in an antioxidant medium, and DA and GABA neurons eval- uated by immunocytochemistry. 6-OHDA induced morpholo- gical 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 lM), 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 apop- tosis, and this effect can be attenuated early but not late by GDNF. Keywords: apoptosis, caspase, neuroprotection, Parkin- son’s disease, trophic factor, ventral tegmental area. J. Neurochem. (2004) 89, 776–787. Parkinson’s disease is characterized by the progressive degeneration of dopamine (DA) neurons in the substantia nigra (SN). Although the cause of this loss is unknown, evidence suggests a role for oxidative stress (see reviews by Olanow and Tatton 1999; Stokes et al. 1999). Thus, an understanding of the mechanisms by which oxidative stress kills DA neurons and how specific agents can attenuate this neurodegeneration may provide insights into the etiology and treatment of the disease. The intracerebral administration of 6-hydroxydopamine (6-OHDA) has been widely used to cause oxidative stress to DA neurons. Under the right conditions, this neurotoxin can cause the selective loss of DA neurons, thereby producing an animal model of certain key aspects of Parkinson’s disease (reviewed in Zigmond and Keefe 1997). 6-OHDA also has been widely used to damage DA neurons invitro. However, unlike the case for invivo models, these in vitro effects of 6-OHDA have never been shown to be restricted to DA neurons. Indeed, in those cases where it has been examined, the invitro effects of 6-OHDA have been observed to be non-specific (Table 1). As a result, it has been difficult to draw conclusions regarding the effects of selective oxidative stress in DA neurons from such studies. Yet, an Received December 31, 2003; revised manuscript received January 14, 2004; accepted January 16, 2004. Address correspondence and reprint requests to Michael J. Zigmond, Department of Neurology, S-526 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA. E-mail: zigmond@pitt.edu 1 The present address of Yun Min Ding is SBRI J-217, MC 2030, Department of Neurology, University of Chicago, Chicago, IL 60637, USA. Abbreviations used: BME, basal medium eagle; DA, dopamine; DAT, dopamine transporter; DETAPAC, diethylenetriaminepentaacetic acid; GABA, gamma-amino butyric acid; GDNF, glial cell line-derived neurotrophic factor; HEPES, N-(2-Hydroxyethyl)piperazine-N¢- (2-ethanesulfonic acid); 6-OHDA, 6-hydroxydopamine; MPTP, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine; PBS, phosphate-buffered saline; SN, substantia nigra; TH, tyrosine hydroxylase; TH + , TH-im- munostained cells; VTA, ventral tegmental area; z-DEVD-fmk, Z-As- p(OCH 3 )-Glu(OCH 3 )-Val-Asp (OCH 3 )-fluoromethylketone. Journal of Neurochemistry , 2004, 89, 776–787 doi:10.1111/j.1471-4159.2004.02415.x 776 Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787