Conditional knockout of NMDA receptors in dopamine neurons prevents nicotine-conditioned place preference

Abstract

Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction.

Figure 1. Generation of the reporter mouse and and histological characterization of Slc6a-cre induced deletion.

(a) Breeding scheme for the production of the dopamine-neuron specific Rosa-stop-lacZ reporter mice. The dopamine neuron-specific Cre line (Slc6a3+/Cre) line was crossed with the rosaxstopxlacZ mic where “x” stands for the loxP sites. Deletion of the floxed stop sequence by cre recmbinase results in lacZ expression driven be the ubiquitously active rosa26 promoter. (b) Low-power (2×) images of immunofluoresence of two different coronal sections for lacZ and TH in the midbrain of the Rosa26-lacZ reporter mice, showing specific gene deletion in the VTA and substantia nigra. (c) High-power (10×) image of double immunofluoresence for TH (shown in green) and Cre recombinase (shown in red) in VTA dopamine neurons of the Slc6a3+/Cre mice. Cre positive neurons pointed by the arrows were also TH positive.

Figure 2. Generation and characterization of the DA-NR1-KO mice.

(a) Breeding scheme for the production of the dopamine-neuron specific NR1 knockout mice. The dopamine neuron-specific Cre line (Slc6a3+/Cre) line was crossed with the floxed NR1 mice for two generations to produce the conditional knockout mice (Slc6a3+/Cre fNR1/fNR1), or DA-NR1-KO for short. Three groups of littermate mice were used as the controls: Slc6a3+/Cre fNR1/+, Slc6a3+/Cre alone, and wild type littermates. The genotyping was done as previously described^7,8. (b) High-power (20×) image of immunofluoresence for Cre recombinase (shown in red) and fluorescent in situ hybridization for NR1 mRNA (shown in green) in VTA dopamine neurons. NR1 expression was abolished in Cre-positive (arrows), but present in TH-negative (arrow heads) neurons in the VTA of DA-NR1-KO mice.

Figure 3. DA-NR1-KO mice display normal locomotor activities in a novel open field.

Locomotor activity was measured as beam breaks in activity chambers (San Diego Instruments, San Diego, CA). Movements including the ambulatory movement, fine movement and rearing activities were scored based on beam break patterns. Mice were placed into standard sized rat cages which they have never been exposed to. Activities were monitored for 120 minutes. There was no difference between genotypes in tested animals' activities. (Post-Hoc test, P>0.05). Data were calculated as Mean ± SEM. ANOVA analysis (either repeated measure ANOVA or Post-Hoc test or Paired-t test, as appropriated) was used to determine the difference among groups.

Figure 4. Selective prevention of nicotine-conditioned place preference in DA-NR1-KO mice.

DA-NR1-KO mice failed to develop nicotine-induced conditioned place preference. The mice from all groups spent roughly 30% of total time in the non-preferred chamber before injection of nicotine or saline. After 4 consecutive days of counterbalanced injections of nicotine in the non-preferred chamber and saline in the preferred chamber, performance differences in genotype and interaction of genotype × day were revealed (Repeated Measure ANOVA, genotype F(3,33) = 5.52, P<0.01; interaction of genotype×day F(3,33) = 13.14, P<0.01). The three control groups of mouse each showed similarly strong preferences to the previously non-preferred chamber (Post-Hoc test, P<0.01 for the three groups, respectively). In contrast, the DA-NR1-KO mice did not show preference to the nicotine paired non-preferred chamber (Paired-t test, P>0.05). Post-hoc test showed significant difference between DA-NR1-KO mice and the control groups (P<0.01, respectively), but no difference among the three control groups. (P>0.05, respectively). Data were calculated as Mean ± SEM. ANOVA analysis (either repeated measure ANOVA or Post-Hoc test or Paired-t test, as appropriated) was used to determine the difference among groups.

Figure 5. Normal air puff-induced conditioned place preference in the DA-NR1-KO mice.

CPP was done using the symmetric conditioning chamber. The mice from all groups spent roughly 50% of total time in the air puff chamber before the air puff was delivered. After air puff (on day 1), when the mice were tested on day 3, the 48-hour retention tests revealed that all mice groups exhibited strong preference to the safe chamber and spent less time in the air puffed chamber (Paired-t test, P<0.01 for the three groups, respectively). Moreover, there was no difference between genotypes in their percentage of time spent in air puffed chamber among all the four groups tested (Post-Hoc test, P>0.05). Nor any difference in interaction of genotype×day was found (Repeated Measure ANOVA, genotype F(3,34) = 1.14, P>0.05; interaction of genotype×day F(3,34) = 0.14, P>0.05 ). Data were calculated as Mean ± SEM. ANOVA analysis (either repeated measure ANOVA or Post-Hoc test or Paired-t test, as appropriated) was used to determine the difference among groups.