NMDA, Sensitization, and VTA Figure 2. Absence of functional NMDAR in NR1DATCre mice. (A) Example waveforms recorded at +50 mV from both wildtype (WT) and NR1DATCre (KO) cells before (black trace) and after AP5 application (grey trace). In WT cells, the NMDA component could be obtained by subtracting the AMPA current remaining after AP5 application from the control current, as indicated. In KO cells, only the AMPA mediated EPSC was observed. The gray block represents the time over which the NMDA current was measured. Scale bars are 20 pA and 20 ms. (B) Average NMDA current measured as described in A. Responses were evoked at holding potentials, beginning at 290 mV in 10 mV increments to +50mV. (C) Decay time constant (tau) of EPSCs measured before and after AP5 in control and KO cells. Following AP5 superfusion, the remaining current in the WT cells exhibited similar decay constants as the KO animals. (D) Mean time course of NMDA currents before and after AP5 application. Following AP5 the WT response amplitude was reduced to the same level observed in the KO cells, thus confirming the lack of active NMDA channels in DA NR1DATCre neurons. doi:10.1371/journal.pone.0012141.g002 The differences in DA release under single pulse and pulse train conditions between WT and NR1DATCre mice suggest that deletion of NMDARs has effects on the dynamic output of DA neurons, and it has been suggested that NMDARs located on DA terminals in the NAc may directly regulate DA release [22]. To evaluate this, we examined the effect of acute NMDA receptor blockade on DA release in NAc slices from WT mice. In contrast to the results obtained with the KO mice, the NMDAR antagonist AP5 (40 mM) had no effect on DA release elicited by single pulse stimulation (Fig. 4B). Furthermore, frequency-response curves of DA release, generated prior to and during AP5 application (Fig. 4C), were not significantly different (two way RM-ANOVA; treatment x frequency, F72, 4 = 1.46, p = 0.2239). Therefore, these data suggest that the differences in DA release observed in the NAc following NR1 deletion reflect compensatory changes in DA release dynamics, rather than the loss of an acute modulation of DA release by NMDARs. Our final FSCV experiments determined whether the loss of NMDAR function in DA neurons can influence the direct effects of cocaine on DA uptake via the DAT in the NAc. Application of cocaine (500 nM) to NAc brain slices significantly enhanced the amplitude of the DA signal and prolonged its time course (increased tau) to the same degree in both WT and NR1DATCre KO slices (Fig. 5). Thus, the direct pharmacological effect of cocaine at the DAT in the NAc was unaltered in NR1DATCre mice. The observed differences in DA release in NR1DATCre KO slices might cause changes in locomotor activity in these mice. Therefore, we monitored their activity levels during a 24-hour PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12141