upright microscope (Zeiss Axioscope, Germany), modified to provide a gradient contrast image utilizing infrared illumination. Putative DA neurons were initially selected for recording by their large cell bodies and the presence of Ih, as originally described by Johnson and North [15]. Each cell was labeled with biocytin (0.125%) and processed for tyrosine hydroxylase (TH) immunohistochemistry to confirm its identity as a DA neuron. Recording electrodes (3–5 MV) were filled with (in mM) CsCl, 130; NaCl, 4; MgCl2, 2; EGTA, 1.1; HEPES, 5; Na2-ATP, 2; Na2- creatine-phosphate, 5; Na3-GTP, 0.6; biocytin, 0.125%, pH 7.2 with KOH. Intracellular recordings were performed using an Axopatch 200B (Axon Instruments, Burlingame, CA). Voltage steps and stimulation protocols were delivered using the Strathclyde electrophysiology software package (WCP, courtesy of Dr. John Dempster, Strathclyde University, Glasgow, UK) and an A/D board (ITC-18, Instrutech Corp., Bellmore, NY) residing in a personal computer. Synaptic currents were evoked by 0.2 ms, constant current pulses delivered every 30s via a bipolar tungsten electrode (FHC, Bowdoinham, ME; 300 mm tip separation), placed anterior to the VTA. Stimulus intensity was adjusted to achieve 30–50% of maximum response amplitude. I–V curves were obtained by varying the holding potential between 290 and +50 mV. NMDA current measurements were made by averaging the current, with respect to baseline, between 30 to 35 ms after stimulation. AP5 was applied in a subset of cells to test for NMDA currents. For LTP experiments, intracellular solution contained (in mM): K-gluconate, 140; KCl, 5; HEPES, 10; EGTA, 0.2; MgCl2, 2; Mg- ATP, 4; Na2-GTP, 0.3; Na2-phosphocreatine, 10; bioctytin, 0.125%; pH 7.2 with KOH. Cells were initially patched in voltage-clamp to test for the presence of I . Following this h confirmation, the recording was switched into current-clamp for the duration of the experiment. To prevent action potentials from occluding stimulus-evoked EPSPs, the cell was manually hyper- polarized to 265mV by injecting negative direct current (,0.05– 0.2 nA) through the pipette. Following at least 10 minutes of stable baseline EPSP recording, LTP was initiated using a spike-timing protocol [8]. Immediately preceding the initiation of the LTP protocol, the holding current was removed to allow the cell to freely fire action potentials. The LTP protocol consisted of 20 bursts of 5 pairings of stimulation and postsynaptic depolarization, with the presynaptic stimulation preceding the depolarization by 5 ms. Each postsynaptic depolar- ization was initiated with current injection (1.5–2 nA) lasting 3 ms. The 5 pairings within a burst were separated by 100 ms (10 Hz), and each of the 20 bursts was separated by 5 sec. Following the last burst, the negative current was again applied to drive the cell back to 265 mV for post-LTP recordings of stimulus-evoked EPSPs. Voltammetry. Carbon fibers (7 mm diameter) were prepared as described previously [14,16]. Voltammetric scan and stimulation timing protocols were performed using a voltammetric amplifier (EVA-8, HEKA Electronic) and PCI- based A/D boards (National Instruments) and custom software (courtesy of Dr. Mark Wightman, Univ. of North Carolina). Scans consisted of sweeps from 20.4 to 1.0 V and back to 20.4 V, at a rate of 400 V/s, and were obtained every 100 ms. A 5 sec. (50 scan) control period preceded each electrically-evoked response, and was used to obtain a background current that was digitally subtracted from the current obtained during the peak of the response. Currents were converted to concentration by in vitro calibration of each electrode against a 1 mM DA standard. All signals used in analyses matched the expected voltammetric profile for DA. Under stereoscopic magnification, carbon fibers were NMDA, Sensitization, and VTA lowered to a depth of ,100 mm in the nucleus accumbens core, generally medial to the anterior commissure. A bipolar stimulating electrode was positioned ,75–100 from the carbon fiber. Constant current pulses (10–1000 mA, 1 ms duration) were delivered between voltammetric scans to elicit dopamine release. 10 pulse trains (400–500 mA, 1 ms, 5–60 Hz) were used to assess the frequency-dependence of DA release. For cocaine and AP5 experiments, responses were obtained every 2 minutes using single pulse stimulation. After obtaining a 10-minute stable baseline response, cocaine (500 nM) or AP5 (40 mM) was superfused into the recording chamber for 10–20 minutes, until a stable drug response was observed. Comparisons were performed by averaging 3–4 responses during the control (pre-drug) and post- drug period. DA uptake was assessed by fitting the decay portion of each signal to a single exponential function. The obtained tau values have previously been demonstrated to be related to the efficiency of DAT-mediated uptake of DA (Vmax/Km), and are sensitive to DAT inhibitors [16]. Behavioral procedures NR1DATCre and WT mice were maintained on a 12 h light-dark schedule (lights on 6:00am). Sensitization in activity chamber. Locomotor activity was measured in activity chambers (Omnitech Electronic Inc, Columbus, OH). The monitor contained 16 horizontal and 8 vertical infrared sensors spaced 2.5 cm apart. Mice were placed in the locomotor chambers and their activity was measured for 60 min. Then the animals were injected with either saline (habituation) or cocaine (sensitization), and the measurements continued for another 60 min. The treatment schedule consisted of injections with saline (habituation) daily for 2 days, followed by cocaine (15 mg/kg, i.p.) daily for 4 days. After the last treatment, mice were kept in the home cage. On day 22 animals were challenged with cocaine (15 mg/kg, i.p.). Homecage sensitization. Mice for each genotype were divided into two groups: The first group received saline injections (i.p.) in the homecage for 6 days, while the second group received 15 mg/kg cocaine i.p. for one day, followed by 20 mg/kg i.p. for 5 consecutive days in the home cage. On day 7, both groups were habituated to the locomotor activity chambers (Omnitech Electronic Inc, Columbus, OH) for 60 min prior to receiving a 15 mg/kg cocaine injection. Locomotor activity was measured during habituation and for 60 min after cocaine injection. Infusion of AP5 in VTA. Mice were implanted with bilateral chronic indwelling guide cannulae aimed 1 mm above the VTA (AP 23.0, ML 60.4, DV 23.5 according to Franklin and Paxinos, [17] that was secured by dental cement. Animals were given at least 2 weeks to recover from surgery before receiving infusions (0.5 ml over 2 min into VTA by pump). The tips of the inner injection cannulae extended 1 mm below the guide cannulae into the injection sites. For the sensitization experiment WT, and NR1DATCre mice were administered four pairs of injections (one intracranial and one i.p.), with one pair given everyday. On each injection day, mice were first habituated to the locomotor chamber for 60 min, before receiving their respective intra cranial injection of AP5 (0.04 mM, 0.5 ml each side; Tocris, Ballwin, MO) or saline (0.5 ml each side), followed by a cocaine i.p. injection (15 mg/kg). Immediately following the i.p, injections, mice were returned to the activity boxes and locomotor activity was recorded for an additional 30 min. Conditioned place preference. The conditioned place preference (CPP) chamber consists of two Plexiglas compartments (23.2612.7612.7 cm) with black walls (Med PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12141