Anatomy Of Dopamine Transportor-Mediated Locomotor And Reward Responses To Cocaine

Part1Validation of the cocaine-insensitive dopamine transporter knock-in mice system and the recombined adeno-associated viral vectors containing wild-type dopamine transporter systemObjectiveTo identify the stability of the behavior effect of the knock-in mice with cocaine-insensitive dopamine transporter (DAT-CI mice) response to cocaine, and confirm the functional efficacy of reintroducing wild-type DAT into the DAT-CI mice brian.MethodsTen adult male DAT-CI mice and ten wild type (WT) mice weighing20-30g were assigned into two groups:DAT-CI group and WT group, a conditioned place-preference (CPP) apparatus was used to measure cocaine-induced reward and hyperlocomotion simultaneously of these two groups mice. The CPP procedure consists of a preconditioning test one day, a cocaine/saline conditioning place phase eight days, and a postconditioning test one day.On the preconditioning test day, the three compartments were made distinct from one another by visual and tactile cues, creating three different "environments". Mice were placed into the center compartment and allowed to explore all three compartments for30min. Time spent in each of the three compartments, as well as the total distance traveled, were automatically recorded by the Anymaze video tracking system. Their preference was defined as the difference in time spent in one side compartment versus the other side. Their unconditioned preference was counterbalanced in each group by designating individual mice to receive cocaine in either their initially preferred or initially non-preferred environment, such that the group bias was minimized. During the conditioning phase, the entire apparatus was configured with only one of the two environmental cue sets. The mice were then administered the treatment (either20mg/kg cocaine or the same volume saline) corresponding with the environment for that day. Immediately after administration, they were placed the environment for30min, and the distance traveled was recorded. On the following day, the opposite agent was administered in the appropriate environment, and these alterations proceed for8days. For the postconditioning test, the mice were tested exactly the same way as on the preconditioning test day. The CPP score is defined as the time spent in the cocaine-paired environment (conditioned stimulus, CS+) during the preference tests, minus the time spent in the saline-paired environment (unconditioned stimulus, CS-). Differences in CPP score during the preconditioning test versus the postconditioning test indicated an effect of the drug. Choose six DAT-CI mice, unilaterally inject the AAV-DATwt in the striatum then sacrificed three of them and perfused with4%paraformaldehyde in order to harvest their brain. Cut the brains coronally by the freezing microtome. Immunohistochemical localization of the injection site was performed on floating sections. The other three of them use the fast scanning cyclic voltammetry (FSCV) measure the dopamine release and reuptake in both side of the striatum. All data were expressed as mean±standard deviation (x±s). Differences between groups were analyzed by two-way ANOVA Analysis followed by Bonferroni’s post hoc test. Simple t-tests were used to analyze the differences in groups. P<0.05was considered to be statistically significant. Data analyses were performed with SPSS19.0software.ResultsCompare with the WT group, DAT-CI group mice do not show hyperlocomotion and CPP effect under the20mg/kg cocaine. The immunohistochemistry show the AAV-DATwt successfully expressed in the target brain region. The FSCV indicates that compare with the un-injected side brain region, the injected side show significant dopamine release peak under the cocaine.Conclusions The AAV system could produce functionally relevant effects on dopamine reuptake in DAT-CI mice and sufficiently enough for confidence in proceeding to behavioral experiments. Part2Restoration of cocaine-induced hyperlocomotion by stereotaxic injuction of rAAV vector containing wild-type dopamine transporter into the dorsal striatum combined lateral nucleus accumbens of adult knock-in mice with a cocaine-insensitive dopamine transporterObjectiveTo determine brian regions in which DAT inhibition by cocaine stimulates locomotor activity and/or produces reward.MethodsThirty adult male knock-in mice witih a cocaine-insensitive transporter (DAT-CI mice) weighing20-30g were randomly assigned into three groups:dorsal striatum combined lateral nucleus accumbens (ICPu) group, medial nucleus accumbens (mNAc) group, DAT-CI group, added wild type (WT) group, four groups totally and10mice per each. Bilateral stereotaxic injection of rAAV vector containing wild-type dopamine transporter (AAV-DATwt) into dorsal striatum combined lateral nucleus accumbens and medial nucleus accumbens of DAT-CI mice were carry out in ICPU group and mNAc group, DAT-CI group and WT group had no injection. Four weeks later, a conditioned place-preference (CPP) apparatus was used to measure cocaine-induced reward and hyperlocomotion stimultaneously of these four groups mice The CPP procedure consists of a preconditioning test one day, a cocaine/saline conditioning place phase eight days, and a postconditioning test one day.On the preconditioning test day, the three compartments were made distinct from one another by visual and tactile cues, creating three different "environments". Mice were placed into the center compartment and allowed to explore all three compartments for30min. Time spent in each of the three compartments, as well as the total distance traveled, were automatically recorded by the Anymaze video tracking system. Their preference was defined as the difference in time spent in one side compartment versus the other side. Their unconditioned preference was counterbalanced in each group by designating individual mice to receive cocaine in either their initially preferred or initially non-preferred environment, such that the group bias was minimized. During the conditioning phase, the entire apparatus was configured with only one of the two environmental cue sets. The mice were then administered the treatment (either20mg/kg cocaine or the same volume saline) corresponding with the environment for that day. Immediately after administration, they were placed the environment for30min, and the distance traveled was recorded. On the following day, the opposite agent was administered in the appropriate environment, and these alterations proceed for8days. For the postconditioning test, the mice were tested exactly the same way as on the preconditioning test day. The CPP score is defined as the time spent in the cocaine-paired environment (conditioned stimulus, CS+) during the preference tests, minus the time spent in the saline-paired environment (unconditioned stimulus, CS-). Differences in CPP score during the preconditioning test versus the postconditioning test indicated an effect of the drug. After the CPP test, mice were sacrificed and perfused with4%paraformaldehyde in order to harvest their brains. Then cut the brains coronally by the freezing microtome. Immunohistochemical localization of the injection site was performed on floating sections. All data were expressed as mean±standard deviation (x±s). Simple t-tests were used to analyze the locomotion and CPP effect of cocaine in each group of mice. P<0.05was considered to be statistically significant. Data analyses were performed with SPSS19.0software.ResultsFor cocaine-induced locomotion behavior, WT group and ICPu group mice display significant locomotor stimulation induced by20mg/kg cocaine, relative to saline, whereas DAT-CI group and mNAc group mice do not. For CPP test, the WT group mice display significantly higher place-preference for the cocaine-paired chamber in the postconditioning test relative to the preconditioning test at20mg/kg, whereas the uninjected DAT-CI mice do not. Meanwhile, injection of AAV-DATwt was not sufficient to restore CPP for any of the two injected DAT-CI cohorts.Conclusions DAT-inhibition in the dorsal striatum combined lateral nucleus accumbens alone is sufficient for cocaine’s locomotor stimulating effect. Whereas multiple dopamine-related brain regions may interact to produce cocaine-induced reward effect. Part3Restoration of cocaine-induced reward effect by stereotaxic injuction of rAAV vector containing wild-type dopamine transporter into the ventral midbrain of adult knock-in mice with a cocaine-insensitive dopamine transporterObjectiveTo dissect out specific brian regions in which DAT inhibition by cocaine produces reward effect.MethodsForty adult male knock-in mice witih a cocaine-insensitive transporter (DAT-CI mice) weighing20-30g were randomly assigned into three groups:ventral midbrain (vMB) group, olfactory tubercle (Tu) group, combined regions (CPU,Acb, Tu, vMB) group, DAT-CI group, added wild type (WT) group, five groups totally and10mice per each. Bilateral stereotaxic injection of rAAV vector containing wild-type dopamine transporter (AAV-DATwt) into ventral midbrain, olfactory tubercle and combined regions of DAT-CI mice were carry out in vMB, Tu group and combined regions group, DAT-CI group and WT group had no injection. Four weeks later, a conditioned place-preference (CPP) apparatus was used to measure cocaine-induced reward and hyperlocomotion simultaneously of these five groups mice. The CPP procedure consists of a preconditioning test one day, a cocaine/saline conditioning place phase eight days, and a postconditioning test one day.On the preconditioning test day, the three compartments were made distinct from one another by visual and tactile cues, creating three different "environments". Mice were placed into the center compartment and allowed to explore all three compartments for30min. Time spent in each of the three compartments, as well as the total distance traveled, were automatically recorded by the Anymaze video tracking system. Their preference was defined as the difference in time spent in one side compartment versus the other side. Their unconditioned preference was counterbalanced in each group by designating individual mice to receive cocaine in either their initially preferred or initially non-preferred environment, such that the group bias was minimized. During the conditioning phase, the entire apparatus was configured with only one of the two environmental cue sets. The mice were then administered the treatment (either20mg/kg cocaine or the same volume saline) corresponding with the environment for that day. Immediately after administration, they were placed the environment for30min, and the distance traveled was recorded. On the following day, the opposite agent was administered in the appropriate environment, and these alterations proceed for8days. For the postconditioning test, the mice were tested exactly the same way as on the preconditioning test day. The CPP score is defined as the time spent in the cocaine-paired environment (conditioned stimulus, CS+) during the preference tests, minus the time spent in the saline-paired environment (unconditioned stimulus, CS-). Differences in CPP score during the preconditioning test versus the postconditioning test indicated an effect of the drug. After the CPP test, mice were sacrificed and perfused with4%paraformaldehyde in order to harvest their brains. Then cut the brains coronally by the freezing microtome. Immunohistochemical localization of the injection site was performed on floating sections. All data were expressed as mean±standard deviation (x±s). Simple t-tests were used to analyze the locomotion and CPP effect of cocaine in each group of mice. P<0.05was considered to be statistically significant. Data analyses were performed with SPSS19.0software.ResultsFor cocaine-induced locomotion behavior, WT group and vMB group, combined group mice display significant locomotor stimulation induced by20mg/kg cocaine, relative to saline, whereas DAT-CI group mice do not. For CPP test, the WT group, vMB group and combined group mice display significantly higher place-preference for the cocaine-paired chamber in the postconditioning test relative to the preconditioning test at20mg/kg, whereas the uninjected DAT-CI mice do not.Conclusions The lack of cocaine stimulation and rewarding effects in DAT-CI mice is not due to adaptive changes but due to the lack of cocaine inhibition of DAT. This strengthens our conclusion that cocaine inhibition of DAT is necessary for the rewarding and stimulating effects of cocaine.