and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey. All efforts were made to minimize animal suffering and to reduce the number of animal used. The experiments were performed on Sprague-Dawley rats aged 15–25 (2061) postnatal (P) days. Slice preparation The midbrain slices were prepared as described previously [23,24]. Animals were anesthetized and then killed by decapita- tion. The brain was removed and a midbrain block (containing the VTA) was isolated. It was glued to the cutting stage of a VF-200 slicer (Precisionary Instruments Inc., Greenville, NC, USA). While the brain was kept in ice-cold glycerol-based artificial cerebrospi- nal fluid (GACSF) – containing 252 mM glycerol, 1.6 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 18 mM NaHCO3, and 11 mM glucose, and oxygenated with 95% O2/5% CO2. 200–250 mm thick slices were cut in the coronal plane. The slices (two per animal) were allowed to recover for at least 1 h in a holding chamber in regular artificial cerebrospinal fluid (ACSF), which has the same composition as GACSF, except that glycerol was replaced with 126 mM NaCl. Electrophysiological recording in midbrain slices Cells in midbrain slices were visualized with an upright microscope (E600FN, Nikon, Tokyo, Japan) and near-infrared illumination. Electrical signals were obtained in whole-cell patch clamp technique with MultiClamp 700 A amplifiers (Molecular Devices Co., Union City, CA, USA), a Digidata 1320 A A/D converter (Molecular Devices Co.) and pCLAMP 9.2 software (Molecular Devices Co.). Data were filtered at 2 kHz and sampled at 5 kHz. The patch electrodes had a resistance of 4–6 MV when filled with the pipette solution containing (in mM) 125 K gluconate, 2.8 NaCl, 2 MgCl2, 0.6 EGTA, 10 HEPES, 2 ATP- Na, and 0.3 GTP-Na for whole-cell current clamp recordings. For whole-cell voltage clamp recordings the patch electrodes were filled with a pipette solution containing (in mM): 135 K gluconate, 5 KCl, 2 MgCl2, 10 HEPES, 2 Mg ATP, 0.2 GTP, and 2 QX- 314. The pH was adjusted to 7.2 with Tris base and osmolarity to 300 mOsmol/L with sucrose. A single slice was transferred into a 0.4 ml recording chamber, where it was held down by a platinum ring. Warm carbogenated ACSF flowed through the bath (1.5– 2.0 ml/min). All recordings were carried out at 32uC, maintained by an automatic temperature controller (Warner Instruments, Hamden, CT). For analyzing the changes in firing rate, we adapted the method used by Brodie’s group [25]. Specifically, firing rate was calculated over 1-min intervals before administra- tion of drugs and during the drug effect; peak drug-induced changes in firing rate were expressed as the percentage change from the control firing rate according to the formula [(FRD- FRC)/FRC] X 100, where FRD is the firing rate during the peak drug effect and FRC is the control firing rate. Thus, the change in firing rate is expressed as a percentage of the initial firing rate, which controls for small changes in firing rate, which may occur over time. All EPSCs were recorded at a holding potential (Vh) of 270 mV. Physiological identification of DA neurons was based on the presence of hyperpolarizing current (Ih), which was greater than 60 pA during a step from 250 to 2100 mV, and the rate of spontaneous action potential activity (1–5 Hz) with spike widths $1.2 ms [21,26,27]. To evoke monosynaptic excitatory postsynaptic currents (eEPSCs), a bipolar stainless steel-stimulating electrode was placed 50–200 mm away from the recorded VTA neuron. Electrical stimuli (100–200 ms in dura- Salsolinol Facilitates Glutamate Transmission tion) were applied at the rate of 0.05–0.1 Hz. Near the start of the recording, an input/output curve was obtained, and the stimulation was then set to 20–30% of the maximum, an intensity that evoked stable responses with no failures. Paired eEPSCs were elicited with a pair of identical stimuli separated by an interval of 50 ms. Series resistance was checked before and after the experiments by series resistance compensation. To measure input resistance, we applied a 5 mV, 400 ms hyperpo- larizing pulse and divided 5 mV by the final value of the evoked current. The data would be discarded if series resistance (15– 30 MV) or input resistance (300–500 MV) changed by more than 20% during the whole-cell recording. Data analysis Spontaneous discharges and excitatory postsynaptic currents (EPSCs) were counted and analyzed with Clampfit 9.2 (Molec- ular Devices Co). Spontaneous or miniature EPSCs (sEPSCs and mEPSCs) were screened automatically (8 pA amplitude thresh- old), checked visually, and accepted or rejected according to their rise (,1.5 ms) and decay (,3.5 ms) times. The height of evoked eEPSCs (eEPSCs) was measured with Clampfit 9.2 and was used to calculate the paired pulse ratio (PPR = EPSC2/EPSC1), where EPSC1 and EPSC2 were evoked by the first and second stimuli at an interval of 50 ms. The eEPSC amplitude, PPR, and the frequency of sEPSCs and mEPSCs during and after drug applications were normalized to their mean value during the initial control period (.3 min). These data were used to depict summarized time courses (10–30 s per/bin). The baseline mean values were obtained during the initial control period and the mean values were obtained during drug application over a 2– 3 min period at the peak of a drug response. Drug effects were expressed as % change (mean 6 SEM) from a pre-drug control baseline. Paired or unpaired two-tailed t-test evaluated the statistical significance of various drug effects (% change from control baseline). Cumulative probability plots of the incidence of various inter-event intervals and amplitudes (for 50–1000 sEPSCs and mEPSCs), were recorded in control conditions and during drug applications to the same neuron, were analyzed with the Kolmogorov-Smirnov (K–S) test. In the figures, single eEPSCs or paired eEPSCs are averages of .10 successive traces. Values of p,0.05 were considered significant. Chemicals and Applications 1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydro- bromide (Salsolinol), bicuculline, DL-2-amino-5-phosphono-vale- ric acid (DL-APV), 6, 7-dinitroquinoxaline-2, 3-dione (DNQX), QX-314, tetrodotoxin (TTX) were obtained from Sigma-Aldrich Chemical Company (St Louis, MO, USA). (6)-SKF 83566 was from TOCRIS Bioscience (Ellisville, MO, USA). The 10006stock solutions were aliquoted and then stored at 220, 4uC, or room temperature according to the catalog recommendations. APV (50 mM) and DNQX (20 mM) were dissolved in ,0.1 N NaOH and other agents in deionized water. Drugs at final concentration were added to the superfusate. Results Salsolinol-induced increase in the firing rate of p-VTA DA neurons is substantially attenuated by antagonists of Gluergic NMDA and AMPA receptors Whole-cell recording was performed in p-VTA-DA neurons in coronal midbrain slices of rats. We chose p-VTA because a previous in vivo study found that salsolinol has an effect on the posterior but not anterior VTA [8]. DA neurons were identified by PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36716