NMDA, Sensitization, and VTA Figure 1. Cre mediated recombination of the NMDAR gene occurs in most ventral tegmental DA neurons with reduced DAT expression levels. (A) Maps showing the endogenous DAT gene (DATwt), and targeted variant DATCre. Black unlabeled boxes represent translated exons of the DAT gene and the light grey box shows the 39UTR. The stop codon has been labeled with an asterisk. A targeting vector containing the Cre recombinase gene (nlsCre) preceded by an IRES (i) was targeted by homologous recombination into the 39UTR of the DAT gene. (B) PCR screen for Cre-mediated Grin1 exon 11–22 deletion. Genomic DNAs were prepared from the indicated tissues from a NR1DATCre mouse. PCR analyses show Grin1 deletion (DloxP) is specific to the olfactory bulb and VTA/SN. Grin1 deletion was not detected in other brain areas or tissues analyzed. (C) Confocal image of DAT-(green) and TH-(red) immunoreactive neurons located in the parabrachial nucleus of the VTA in a NR1DATCre animal. TH immunoreactive neuron pointed with a white arrow does not show detectable DAT immunoreactivity. TH immunoreactive neurons (2–5 neurons per sample) with undetectable (small arrow, n = 36 samples) and detectable DAT levels (n = 15 samples) were collected by laser-microdissection for DNA extraction and PCR processing. Scale bar = 30 mm. (D) PCR analyses of laser-microdissected neurons from NR1DATCre animals revealed Cre mediated recombination in DA neurons with both, low and high DAT protein levels. DNA extracted from tissue surrounding DA neurons within the VTA did not recombine at the NMDAR gene locus. doi:10.1371/journal.pone.0012141.g001 Altered glutamate signaling in DA neurons results in altered DA release in the nucleus accumbens and abnormal locomotor behavior Previous studies suggest that DA release may be altered in DA neuron target regions following loss of NMDARs [10,11,21]. To assess the dynamics of DA release in the NAc, fast-scan cyclic voltammetric (FSCV) recordings were performed in brain slices obtained from either NR1DATCre mice or WT controls. We first compared the relationship between stimulus intensity and DA release, using local single pulse electrical stimulation. This stimulation paradigm releases lower levels of DA, and may therefore approximate the ‘tonic’ release of DA seen when DA neurons fire at low frequencies. NR1DATCre mice showed a slight but significant enhancement of DA release compared to control mice (Fig. 4A), an effect that was most apparent at higher stimulus intensities. A two-way repeated measures ANOVA revealed a significant interaction between genotype and stimulus intensity (F440,10 = 1.91, p = 0.04). This effect was not due to a change in DA uptake, as evidenced by the finding that the decay time constants of the DA signals were unchanged in NR1DATCre mice, relative to WT controls (unpaired two-tailed t-test, p = 0.2491). We next assessed whether NR1 deletion in DA neurons might alter DA release under conditions in which DA neurons would be more active using 10 pulse trains of stimuli, delivered at varying frequencies. Such release may correspond to ‘phasic’ DA release elicited during repetitive (‘‘burst’’) firing of DA neurons. In contrast to the enhanced release observed under single pulse conditions, we found that brain slices from NR1DATCre mice showed a significant decrease in frequency-dependent, DA release (Figure 4C, two way RM-ANOVA, genotype x frequency, F160,4 = 3.57, p = 0.008). PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12141