NMDA, Sensitization, and VTA Figure 7. Cocaine induced sensitization in NR1DATCre mice. (A) Context dependent sensitization. Cumulative 30 min locomotor re- sponse to cocaine (15 mg/kg, i.p.) in WT (n = 7) and NR1DATCre (n = 8) animals at different days after injection. S2, day 2 of saline injection. C4, day 4 of cocaine injection. C20 represents a challenge cocaine injection after withdrawal. Cocaine exposure in association with a new environ- ment induced significant sensitization in both WT and NR1DATCre mice (two way ANOVA, Newman-Keuls post hoc for all genotypes: *p,0.05 for WT compared to saline injection, ** p,0.01 for KO compared to saline injection; #p,0.05 for WT compared to C1, ## p,0.01 for KO compared to C1). No statistical differences were observed between genotypes neither at the initial development of sensitization (C1 versus C4) nor at the challenge injection after withdrawal (C20, two way RM- ANOVA, genotypes, F1, 65 = 0.00703, p = 0.934). (B) Cocaine-induced sensitization in the home cage. Cumulative 30 min locomotor response to cocaine in WT and NR1DATCre animals pretreated with saline (WT, n = 8 and KO, n = 6) or 20 mg/kg cocaine, i.p. (WT, n = 8 and KO, n = 7) in the home cage, prior to cocaine challenge (15 mg/kg, i.p.) in activity chambers. Cocaine exposure in the home cage induced significant sensitization in both, WT and NR1DATCre mice (Two way ANOVA, F1,28 = 3.57, p,0.001; Newman-Keuls post hoc for all genotypes: * p,0.05 for KO, ** p,0.01 for WT). Data presented as mean 6 SEM. doi:10.1371/journal.pone.0012141.g007 no significant difference between times spent on the drug-paired floor during pre-test and reinstatement-test. However, there was significant difference between saline- and cocaine-paired com- partment for each genotype (F(1,15) = 7.47; p = 0. 016 and F(1,15) = 4.85; p = 0. 045, for WT and NR1DATCre animals, respectively). Discussion The involvement of local glutamatergic synapses within the VTA in learning and the development of adaptive processes in animal models of drug abuse has been widely documented [25]. The fact that both, single or repeated administration of an Figure 8. Effect of pretreatment with AP5 in the VTA on the development of behavioral sensitization to cocaine in NR1DATCre mice. (A) Location of the guide cannula and the injection cannula in the ventral tegmental area (VTA). Left panel is the illustration from the mouse brain atlas [17], indicating millimeters from bregma. The right half shows the image with dyes indicating the position of cannula tips. (B) Preceding cocaine injections with intra-VTA infusions of AP5 blocks the induction of locomotor sensitization by cocaine in both WT (n = 8) and NR1DATCre (n = 5) mice, an effect that was not observed by intra-VTA saline injections in WT (n = 5) and NR1DATCre (n = 6). Data were collected on days 1 and 4 of cocaine treatment. The results show a significant difference between days 1 and 4 for the saline-infused animals (* p,0.001, two way ANOVA, day, F1,21 = 18.533), but not for those treated with intra-VTA AP5 (p = 0.679, two way ANOVA). doi:10.1371/journal.pone.0012141.g008 addictive drug directly into the VTA initiates sensitization to subsequent systemic drug challenge, together with the observation that NMDAR antagonists delivered directly to the VTA block the development of sensitization [1,4,5], suggests a role for glutama- tergic transmission within the VTA for the initiation of addictive behaviors. However, the precise substrates through which altered glutamatergic transmission results in drug modified synaptic plasticity and the development of addictive behaviors remain to be elucidated. Our results extend previous findings by using a transgenic mouse lacking the NR1 receptor subunit in DA neurons (NR1DATCre), and by confirming the absence of NMDARs in virtually all VTA DA neurons. Since our approach, like previous studies [6,10,11,21], relied upon DAT expression for the selective elimination of NMDAR function in DA neurons, we considered it important to determine whether NR1 gene deletion occurred in mesostriatal and mesocortical circuits, because the latter is PLoS ONE | www.plosone.org 10 August 2010 | Volume 5 | Issue 8 | e12141