Acute effects of 6-hydroxydopamine on dopaminergic neurons of the rat substantia nigra pars compacta in vitro.
6-Hydroxydopamine (6-OHDA) is a neurotoxin which has been implicated in the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD), and is frequently used to produce animal models of the disease. The aim of our study, conducted on midbrain slices obtained from young Wistar rats, was to determine the little known acute effects of this toxin (0.2-2.0 mM; 10-20 min exposure; 34 degrees C) on electrophysiological properties, intracellular Ca2+ levels and dendritic morphology of SNc neurons. Four experimental approaches were used: extracellular recording of firing frequency, whole-cell patch-clamping, ratiometric fura-2 imaging, and cell labeling with lucifer yellow (LY) or dextran-rhodamine. Extracellular recording revealed a concentration-dependent decrease in the tonic, pacemaker-like firing. In whole-cell recordings in voltage-clamp (V(hold) -60 mV), smaller doses (0.2-0.5 mM) induced an outward current (or cell membrane hyperpolarization in current-clamp), which could in some cells be reversed with tolbutamide (blocker of ATP-dependent K+ channels). A higher dose (1.0-2.0 mM) caused rapid reductions of cell membrane capacitance and membrane resistance. Toxin exposure gradually increased the intracellular Ca2+ level, which did not subsequently return to control. The increase in Ca2+ signal was not prevented by depletion of intracellular Ca2+ stores with thapsigargin (10 microM) or cyclopiazonic acid (30 microM), nor by removing extracellular Ca2+. Cell membrane current and Ca2+ responses were not prevented by blocking dopamine transporter (DAT). Cells loaded with LY or dextran-rhodamine showed signs of damage (cell membrane blebbing) in dendrites following toxin exposure (1 mM; 10-20 min). These results demonstrate that the oxidative and metabolic stress induced in SNc neurons by 6-OHDA results in rapid dose-dependent changes of cell membrane properties with morphological evidence of dendritic damage, as well as in disturbance of intracellular Ca2+ homeostasis.
Berretta
Nicola
N
Laboratory of Experimental Neurology, Fondazione Santa Lucia IRCCS, Via Ardeatina 306, 00179 Rome, Italy. n.berretta@hsantalucia.it
Freestone
Peter S
PS
Guatteo
Ezia
E
de Castro
Denis
D
Geracitano
Raffaella
R
Bernardi
Giorgio
G
Mercuri
Nicola B
NB
Lipski
Janusz
J
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D003712:Dendrites / Q000187:drug effects / Q000473:pathology; D004298:Dopamine / Q000502:physiology*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors; D004594:Electrophysiology; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007546:Isoquinolines; D008564:Membrane Potentials / Q000187:drug effects; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects*; D016627:Oxidopamine / Q000633:toxicity*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013378:Substantia Nigra / Q000502:physiology*
D050483:Dopamine Plasma Membrane Transport Proteins; D005456:Fluorescent Dyes; D007546:Isoquinolines; D012235:Rhodamines; D008734:Methoxyhydroxyphenylglycol; D016627:Oxidopamine; C017475:lucifer yellow; C010117:3,4-dihydroxyphenylglycol; D004298:Dopamine