S., Guillot T. 30% reduction of MPP+-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the power of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP+ on neuronal DA homeostasis and neurotoxicity. (33,C35). However, many of the proposed effects of MPP+ have never been exhibited experimentally. We studied the time and concentration dependences of the alterations in DA metabolic pools in MPP+-treated acute striatal slices and primary cultures of midbrain dopaminergic neurons. Our findings indicate that MPP+ affects DA vesicular storage, DAT-mediated transport, and catabolic breakdown, leading to the accumulation of DAcyt and neurotoxicity. EXPERIMENTAL PROCEDURES Animals C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were used for slice preparations, and Tamsulosin hydrochloride ventral midbrain primary cultures were used for neurotoxicity and HPLC experiments. For intracellular patch electrochemistry (IPE), cultures were generated from transgenic mice that express GFP under Tamsulosin hydrochloride the control of the rat tyrosine hydroxylase (TH) promoter (TH-GFP+/?) (36). VMAT2 overexpression experiments were performed on ventral midbrain neurons from Sprague-Dawley rats. All animals were used in accordance with the National Institutes of Health guidelines for the use of live animals, and the animal Hpt protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Acute Striatal Slice Preparation and Measurement of DA Release by Fast-scan Cyclic Voltammetry 7C9-week-old mice were decapitated, and 300-m coronal slices that contained cortex and striatum were cut on a Leica VT1200 vibratome (Leica Biosystems Nussloch GmbH, Nussloch, Germany) in ice-cold cutting saline made up of 125 mm NaCl, 2.5 mm KCl, 26 mm NaHCO3, 0.3 mm KH2PO4, 3.3 mm MgSO4, 0.8 mm NaH2PO4, and 10 mm glucose (pH 7.2C7.4, 292C296 mosmol/liter). Slices were allowed to recover for 1C2 h at 37 C in oxygenated (95% O2, 5% CO2) recording saline made up of 125 mm NaCl, 2.5 mm KCl, 26 mm NaHCO3, 0.3 mm KH2PO4, 2.4 mm CaCl2, 1.3 mm MgSO4, 0.8 mm NaH2PO4, and 10 mm glucose (pH 7.2C7.4, 292C296 mosmol/liter). Fast-scan cyclic voltammetry recordings Tamsulosin hydrochloride were performed with 5-m cylinder carbon fiber electrodes (CFEs) positioned at the dorsolateral striatum 50 m below the uncovered surface. Striatal slices were electrically stimulated using a bipolar stainless steel electrode placed at a distance of 100 m from the recording electrode. Square pulses of 0.4-ms duration were produced by an ISO-Flex stimulus isolator triggered by a Grasp-8 pulse generator (A.M.P.I., Jerusalem, Israel). Stimulus magnitude was selected by plotting a current-response curve and selecting the minimum value that produced the maximum response. Triangular voltage ramps from a holding potential of ?450 mV to +800 mV over 8.5 ms (scan rate of 295 mV/ms) were applied to the CFEs at 100-ms intervals. Current was recorded with an Axopatch 200B amplifier (Molecular Devices, Foster City, CA) filtered with a 10-kHz low-pass Bessel filter and digitized at 25 kHz (ITC-18 board, InstruTECH, Great Neck, NY). Triangular wave generation and data acquisition were controlled by a locally written computer routine in IGOR Pro (WaveMetrics, Lake Oswego, OR). Background-subtracted cyclic voltammograms obtained in DA solutions of known concentration served to calibrate the electrodes and to identify released DA. Cell Cultures Ventral midbrain dopaminergic neurons from postnatal day 0C2 mice were dissected, dissociated, and plated on a monolayer of cortical astrocytes at a plating density of 100,000 cells/cm2 as described (37, 38). Experiments were conducted 7C14 days post-plating. Adenoviral Vector Construction and Transfection HA-tagged VMAT2 cDNA was first subcloned into the shuttle vector pTet-EF and then co-infected with donor computer virus DNA (5) into HEK293 cells expressing Cre recombinase, and the resultant VMAT2-HA-harboring adenovirus was purified and stored at 8000 pfu/l as described previously (39). Rat primary ventral midbrain.