NMDA, Sensitization, and VTA Figure 9. Development of cocaine-induced CPP, extinction, and reinstatement. NR1DATCre (n = 8) and WT (n = 8) animals exhibit no significant difference between saline and cocaine-associated compartments before conditioning (A, pre-test) and significant conditioned place preference for the cocaine-associated compartment following conditioning (B). The data are expressed as the time in seconds spent in the saline and cocaine paired compartments during the pre-test (A), preference test (B), after extinction (C), and reinstatement (D) for WT and KO animals 6 SEM. Cocaine dose was 10 mg/kg. Significant differences were found in time spent in cocaine-paired compartment as compared to pre-test (*), and time spent in cocaine-paired compartment as compared to saline-paired compartment within each test session (#). Two-factor repeated measures ANOVA, p#0.05. doi:10.1371/journal.pone.0012141.g009 characterized by DA neurons with low or undetectable DAT protein levels [12]. It has been speculated that remaining NMDAR function in these neuronal circuits might account for the initiation of behavioral sensitization in NR1 KO animals [26]. Our study provides the first direct evidence that the NR1 coding region in VTA DA neurons of NR1DATCre mice is absent in nearly all DA neurons, even those with undetectable or low DAT protein levels. This suggests that the low activity of the DAT promoter in these cells is sufficient to drive Cre-mediated recombination. Alternatively, a transient elevation of DAT expression levels during development [27] may have induced the permanent deletion of NR1 that we observe in adult NR1DATCre animals. Although NMDAR function was disrupted in virtually all DA neurons in our NR1DATCre transgenic mice, these animals demonstrated normal sensitization to repeated cocaine treatments in both, the home cage and a novel environment. Thus, these data agree with previous findings [10,11]. However, other behavioral landmarks of addictive behavior do not correlate well among these studies. For example, Zweifel et al. [11,21] reported a lack of CPP in NR1 KO animals, whereas CPP was intact in the study by Engblom et al, [10], and in our NR1DATCre animals. In addition, we failed to observe differences between NR1DATCre and WT mice with regard to long-term changes in cocaine sensitivity following sensitization [11], and to reinstatement of CPP following extinction [10]. Although a ready explanation for these inconsis- tencies is presently lacking, it is possible that differences in phenotype of the distinct transgenic lines of mice used in these experiments is responsible. For example, the strategy used by Zweifel et al. [11,21] eliminated one of the two alleles at both, the Slc6a3 and Grin1 locus, except in DA neurons where Cre mediated recombination eliminated the remaining Grin1 allele. Therefore, these mice may exhibit an unexpected behavioral phenotype resulting from the partial reductions of DAT and NR1 expression levels. In contrast, Engblom et al. [10] used a bacterial artificial chromosome (BAC)-DAT transgene to drive Cre recombinase expression in DA neurons, and control animals did not contain this transgene [10]. This could be problematic because the random integration site of the BAC transgene may alone affect cellular function [28], and the number of integrated copies determines Cre recombinase expression levels in neurons with low and high DAT levels [29]. In addition to these issues with transgenic constructs, genetic background may also impose variability in behavioral studies [30]. In contrast to these approaches, we targeted the stop codon at the DAT gene locus to drive Cre recombinase expression with help of an internal ribosome entry site (IRES) sequence. This strategy preserved DAT expression through both Slc6a3 alleles, and eliminated potential problems derived from the use of BAC transgenic animals [28]. The normal progression of cocaine sensitization in NR1 KO mice in this, and in previous studies was generally unexpected because it was thought that NMDARs on VTA DA neurons were necessary to support this response. Previous work suggested that the persistence of sensitization in these KO mice may result from the elimination of this gene in only those DA neurons that express moderately high levels of DAT, since this promoter was used to drive Cre recombinase. However, another explanation is needed to explain the persistence of cocaine sensitization in these transgenic mice because we found that the NR1 gene was eliminated in virtually all DA neurons examined. This observation, together with our demonstration that intra-VTA infusions of an NMDAR antagonist fully block sensitization in NR1DATCre mice, suggest that although functional VTA NMDARs are required for cocaine sensitization, the location of the relevant receptors must be on non-DA neurons in the VTA. Despite our data supporting the idea that it is NMDARs on non-DA neurons in the VTA that are critical for the initiation of PLoS ONE | www.plosone.org 11 August 2010 | Volume 5 | Issue 8 | e12141