Acute Effects of 6-Hydroxydopamine on Dopaminergic Neurons of the Rat Substantia Nigra Pars Compacta In Vitro Nicola Berretta 1,y, * , Peter S. Freestone 3,y , Ezia Guatteo 1 , Denis de Castro 3 , Raffaella Geracitano 1 , Giorgio Bernardi 1,2 , Nicola B. Mercuri 1,2 , Janusz Lipski 3 1 Laboratory of Experimental Neurology, Fondazione Santa Lucia IRCCS, Via Ardeatina 306, 00179 Rome, Italy 2 Department of Neuroscience, University of Rome-Tor Vergata, Rome, Italy 3 Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand Received 10 December 2004; accepted 10 January 2005 Available online 10 May 2005 Abstract 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 8C) on electrophysiological properties, intracellular Ca 2+ 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 exposuregradually increased the intracellular Ca 2+ level, which did not subsequently return to control. The increase in Ca 2+ signal was not prevented by depletion of intracellular Ca 2+ stores with thapsigargin (10 mM) or cyclopiazonic acid (30 mM), nor by removing extracellular Ca 2+ . Cell membrane current and Ca 2+ 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 Ca 2+ homeostasis. # 2005 Elsevier Inc. All rights reserved. Keywords: Substantia nigra; Dopamine neurons; Brain slice; 6-OHDA; Parkinson’s disease INTRODUCTION The main pathological characteristic of Parkinson’s disease (PD) is the progressive degeneration of striatum- projecting dopamine (DA) neurons in the substantia nigra pars compacta (SNc), which leads to a debilitating motor dysfunction. The etiology of PD remains obscure, but both environmental and genetic factors have been suggested to play a role (Schapira, 1997; von Bohlen und Halbach et al., 2004). Involvement of epigenetic factors in DA cell death has been suggested following the observation in the early 1980s that 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) produced NeuroToxicology 26 (2005) 869–881 * Corresponding author. Tel.: +39 06 5150 1382; fax: +39 06 5150 1384. E-mail address: n.berretta@hsantalucia.it (N. Berretta). y Berretta and Freestone contributed equally to this work. 0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.01.014