Associates Inc, St. Albans, VT, USA). The CPP procedure consisted of four phases: habituation/pre-conditioning, conditioning, preference test, and extinction/reinstatement. CPP was measured as amount of time spent in cocaine-paired compartment. On day one (habituation), both compartments of the CPP chamber were equipped with the same floor type (smooth plastic surface) and animals were allowed to explore both compartments during one session of 15 min. Next day, for pre- conditioning (pre-test), one compartment was equipped with a mesh floor and the other compartment with grid-mesh floor. Mice were allowed to explore both compartments during one session of 15 minutes. Animals that spent more than 65% of the time in any one compartment were eliminated from the experiment. Overall CPP conditions were unbiased for both phenotypes. The next eight days were dedicated to the conditioning phase. During the conditioning phase subjects had access to the entire apparatus and pairing of saline or cocaine with a particular floor type was randomly assigned in each group. Animals received one conditioning trial each day after an alternative injection of saline or cocaine (10 mg/kg i.p.). Immediately after the injection, mice were placed in the conditioning apparatus containing the corresponding stimulus floor type and were allowed to explore both compartments during 30 min. The presentation order of saline and cocaine and floor- pairing was counterbalanced within and between genotypes. The preference test took place on day 9, or 24 hr after the last conditioning trial. To conduct this test, the chambers were equipped with half mesh and half mesh-grid floor, and mice were placed in the chamber without any previous injection. The preference test duration was 15 min, and the time spent on the floor paired with cocaine and saline was measured. The primary index for deciding whether place conditioning has occurred was significant difference between times spent on the drug-paired floor during the pre-test and the preference test. Extinction consisted of 8 daily sessions in which both compart- ments were equipped with the same floor distribution as during the preference test (half mesh and half mesh-grid floor). Mice were allowed to explore both compartments during one daily session of 30 min. On day 18, for the extinction test, compartments were equipped with the same floor as during the preference test, and mice were allowed to explore both compartments during one session of 15 min. On day 19, for the reinstatement test, the same floor order was used. Animals received a single cocaine injection (10 mg/kg, ip) and were allowed to explore both compartments during one session of 15 min. Results Cre mediated recombination is restricted to the ventral mesencephalon and olfactory bulb in NR1DATCre mice To assess the spatial specificity of Cre mediated recombination in NR1DATCre animals, we examined recombination at the Grin1 gene locus in different organs of the mouse using PCR primers specific to the recombined DNA (Fig. 1B). PCR analyses showed Cre mediated recombination in the VTA, substantia nigra (SN), and olfactory bulb. All other areas analyzed did not show the recombination band (Fig. 1B). VTA DA neurons with low or undetectable DAT protein levels express Cre mediated recombination It has recently been demonstrated that the adult dopaminergic system is composed of two functionally and molecularly distinct types of DA neurons with anatomical segregation in the midbrain, and with non-overlaping axonal targets [12]. Mesocorticolimbic midbrain DA neurons projecting to the medial prefrontal cortex NMDA, Sensitization, and VTA and basolateral amygdala fire action potentials at significantly higher frequencies and posses significantly lower DAT mRNA expression levels, compared to other DA subtypes [12]. It has been suggested that mesocorticolimbic DA neurons with low DA reuptake capacity may mediate sustained forms of behaviorally relevant DA release in vivo [18,19,20]. Using antibodies against TH and DAT we identified many VTA DA neurons with low or undetectable DAT expression levels (Fig. 1C), mostly clustered in the medial posterior VTA, and predominantly located in the medial aspect of the paranigral and parabrachial nucleus, as previously described [12]. To address the question of whether DA neurons with undetectable DAT levels express Cre mediated recombination in NR1DATCre animals, we collected populations of TH immunore- active neurons in the VTA that lacked DAT immunoreactivity using UV-laser microdissection, and analyzed them using PCR (Fig. 1C). For comparison, neurons that were labeled by both TH and DAT antibodies, and non-immunoreactive tissue within the VTA, were also collected and analyzed by PCR. Selected pools of 2–5 labeled neurons each, or non-immunoreactive VTA tissues, were collected from coronal fixed cryosections. Primers specific for the recombination event in the Grin1 gene locus were utilized to ascertain if VTA DA neurons express Cre mediated recombina- tion. Thirty-four of thirty-six samples containing TH immunore- active/DAT negative neurons expressed a robust recombination band, indicating Cre mediated recombination (Fig. 1D). All samples with TH/DAT double stained neurons showed the recombination band (n = 15; Fig. 1D). Non-immunoreactive tissue surrounding VTA DA cells did not show the recombination band (n = 5; Fig. 1D). Thus, contrary to previous studies suggesting the absence of recombination in low-DAT expressing DA neuron subpopulations, our PCR analyses suggests that DNA coding for the NR1 subunit had recombined in nearly all of these neurons. Inactivation of NMDARs in DA neurons eliminates the induction of synaptic strengthening To test whether or not the selective deletion of the NR1 subunit resulted in functional inactivation of the NMDA channel in VTA DA neurons, we performed intracellular recordings of evoked EPSCs in brain slices containing the VTA. At a holding potential of +50mV, neurons from WT animals displayed large NMDA currents (,100 pA), whereas no NMDA currents were seen in DA neurons from NR1DATCre mice (Fig. 2A–B). We also observed large NMDA currents in non-DA VTA neurons in the NR1DATCre animals, confirming the selectivity of the KO (n = 4, data not shown). To further verify the absence of an NMDA component in the synaptic responses in KO animals, we applied the selective NMDAR antagonist AP5. This antagonist did not alter EPSCs evoked in NR1DATCre DA cells, but it did reduce the amplitudes of EPSCs evoked in control cells (Fig. 2C–D). Furthermore, the remaining current following AP5 application was completely blocked by the AMPA receptor antagonist, DNQX in both groups of cells (data not shown). The activation of NMDARs on VTA DA neurons by endogenous glutamate has previously been established as neces- sary for the expression of LTP in these cells [9]. In current clamp recordings, a spike-timing dependent LTP protocol [8] reliably generated LTP in cells from WT animals, but was never observed in VTA DA neurons from NR1DATCre animals (Fig. 3B), thereby confirming that functional NMDARs are required for VTA DA cells to express this form of LTP. During recordings, all cells were filled with biocytin, and subsequently processed for the immuno- histochemical presence of TH (Fig. 3A). PLoS ONE | www.plosone.org 4 August 2010 | Volume 5 | Issue 8 | e12141