Engblom et al., [10] found that CPP was unaltered in similar KO mice. However, despite these discordant observations, both groups reported that DA neuron NR1 deletion altered long-term behavioral plasticity determined using cocaine challenge injections in previously sensitized mice [11], and CPP reinstatement following extinction [10]. The absence of effects of DA neuron NR1 KO on the development of cocaine sensitization and CPP has led to the proposal that NMDARs remaining on mesocortical and mesoa- mygdala projecting DA neurons may be those that are critical for these cocaine-associated phenomena. This is because these populations of DA neurons express little or no detectable levels of DAT [12], and since both studies used a strategy that relied upon DAT expression to target the NR1 KOs, these DA neurons may exhibit relatively normal levels of NMDAR function [10,11]. To further examine the role played by NMDARs in the development of addiction, and specifically its association to the mesocorticolimbic DA system, we developed an independent transgenic line in which the gene for NR1 was deleted in DA neurons by expressing Cre recombinase after the stop codon of the DAT gene locus. We find that the NMDAR coding region is deleted, even in DA neurons with low or undetectable DAT protein levels, that the development of sensitization and CPP progressed normally, despite the absence of NMDAR-dependent LTP in these NR1DATCre mice, and that local intra-VTA application of an NMDAR antagonist blocked behavioral sensitization in the NR1 KO mice. Materials and Methods Mice All animal protocols were conducted under National Institutes Health (NIH) Guidelines using the NIH handbook Animals in Research and were approved by the Institutional Animal Care and Use Committee (National Institute on Drug Abuse, Intramural Research Program, Baltimore, MD), approval ID 09-CNRB-57. Grin1loxP mice were obtained from Jackson laboratories [13]. For the conditional inactivation of Grin1, a transgenic line, Slc6a3Cre, was used in which Cre recombinase is driven by the DA transporter promoter [6]. To minimize interference with gene function by preservation of both alleles, Cre recombinase expression was driven from the 39-untranslated region (39UTR) of the endogenous DAT gene by means of an IRES sequence. The Grin1loxP line [13] was mixed with the Slc6a3Cre to obtain regional knockout (Slc6a3Cre/wtGrin1loxP/loxP, or NR1DATCre) and control mice (Slc6a3Cre/wtGrin1 wt/wt, or WT). Animals were genotyped using Grin1loxP and Slc6a3Cre primers described elsewhere [6,13]. To determine if Grin1 deletion was specific to DAT expressing regions, primers specific to the recombination (DloxP) were utilized. Dissections of the olfactory bulb, ventral mesencephalon, motor cortex, dorsal striatum, lung, heart, kidney, liver and tail were performed and DNA was extracted for PCR analysis. Experiments in this study were conducted in NR1DATCre animals backcrossed in c57bl/6 for at least 6 generations. However, behavioral sensitization (excluding those animals injected with AP5), and CPP experiments were conducted on NR1DATCre and WT animals in congenic c57bl/6 background. All animals used in behavioral studies and voltammetric recordings were between 2–4 months of age; electrophysiological studies utilized mice between 21–30d. Immunohistochemistry and confocal microscopy Proteins were detected with primary antibodies to TH and DAT (Chemicon, CA). Primary antibodies were detected using NMDA, Sensitization, and VTA Alexa Flour 549 and Alexa Flour 488-conjugated, anti-rabbit and anti- rat antibodies (Invitrogene, Carlsbad, CA). Images were analyzed on a Nikon (Melville, NY) Diaphot inverted confocal microscope using the Volocity confocal imaging system (Perkin Elmer). UV-laser microdisection, DNA extraction and PCR analysis Coronal cryosections (5 mm) of fresh frozen NR1DATCre mouse midbrains were cut (Leica cryostat CM3050S), mounted on polyethylene naphthalate (PEN) membrane slides (Leica Micro- systems), fixed with 75% ethanol for 10 min, acetone for 2 min, and air-dried. Sections were incubated overnight at 4uC with antibodies against DAT (1:30,Chemicon, CA) and TH (1:40 Chemicon, CA) in blocking media. Sections were washed with PB and incubated for 3 hours at RT in a mixture of secondary antibodies (Invitrogene, Carlsbad, CA), Alexa Flour 549 anti- rabbit IgG, and Alexa Flour 488 anti-rat IgG, made up in blocking media. Sections were washed with distilled H2O, and air-dried. Fluorescent-labeled neurons were collected under epifluorescent optics using an LMD6000 system. The following types of neurons and tissue within the VTA were collected for PCR analyses: 1) TH and DAT double immunoreactive neurons (n = 15 samples, with 2–5 cells per sample), 2) TH immunoreactive neurons with undetectable DAT expression levels (n = 36 samples, with 2–5 cells per sample), and 3) tissue surrounding TH and/or DAT positive neurons (n = 5 samples). Isolated neurons and tissue were collected by gravity directly into a cap of a 0.5 ml thin-walled PCR-tube. Cell lysis buffer (5 ml per sample) was added directly into the lid, and lysis was performed at 65uC for 2 hours, followed by 10 min at 95uC to inactivate proteinase K. A multiplex reaction for the recombination band, and the control gene GAPDH was performed for each sample. The recombination band (DloxP) was genotyped by PCR primers forward, 59- agatacaagaccctgact- 39 flanking the 59 loxP site, and reverse, 59-cacttgagtagcgccaagtgc-39 in the Pgk promoter of the Pgk-Neomycin cassette, and was sequenced verified. The GAPDH gene was amplified with primers forward, 59- atggtgaaggtcggtgtga -39 and reverse, 59- aatctccactttgcca- ctgc -39. Recordings in brain slices Slice preparation. Animals were rapidly decapitated using a guillotine and their brains were removed and transferred to a beaker containing oxygenated (95% O2/5% CO2), ice-cold artificial cerebral spinal fluid (aCSF) (in mM: sucrose, 194; NaCl, 30; KCl, 4.5; MgCl2, 1; NaH2PO4,1.2; glucose, 10; NaHCO3, 26). Coronal hemisections (280 mm) containing the nucleus accumbens (NAc) were prepared as described previously [14]. NAc slices were were transferred to an oxygenated holding chamber (35uC) filled with normal aCSF (in mM: NaCl, 126; KCl, 3; MgCl2, 1.5; CaCl2, 2.4; NaH2PO4, 1.2; glucose, 11; NaHCO3, 26) for 25–30 min, then gradually cooled to room temperature for $30 min prior to recording. During voltammetric recordings, slices were continuously superfused with aCSF (2 ml/min) and maintained at 31–33uC. For VTA slices, the brain was then blocked to isolate the VTA before being glued onto the cutting stage of a vibrating tissue slicer (Leica, VT1000) and submersed in ice-cold, oxygenated aCSF. Four sagittal slices (280 mm) containing the VTA were obtained from each mouse. The slices were transferred to an oxygenated holding chamber (31uC) filled with normal aCSF and allowed to recover for at least 1 hour before recording. Electrophysiology. One brain slice was transferred to a heated chamber (31–33uC) and superfused (2 ml/min) with aCSF +100 mM picrotoxin. VTA neurons were visualized with an PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12141