The dopamine transporter is a key component in the regulation of DA neurotransmission. Most evidence suggests that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate the stimulating and rewarding effects of cocaine. However, the persistence of the rewarding effect of cocaine in DAT-KO mice suggests a mechanism of DAT-independent cocaine reward, which might be due to the extensive adaptive changes in DAT-KO mice. To test whether the DAT-independent cocaine reward also plays a role in normal mice, we made a knock-in mouse line with the endogenous DAT replaced by a cocaine-resistant DAT mutant. In these DAT-CI mice, cocaine suppresses locomotion and does not produce reward, supporting the hypothesis that cocaine blockade of DAT is required for cocaine reward in normal mice [17]. However, DAT activity in DAT-CI mice is significantly lower than that in wild type mice, and changes in DAT have been shown to alter behaviors or drug responses. For example, reduced DAT activity in DAT-KD mice has been shown to alter behavioral responses to amphetamine, another psychostimulant that impacts the dopaminergic system [18]. It remains a concern that in DAT-CI mice the lack of cocaine response might be due to compensatory changes from the lowered DAT activity. Therefore, we studied cocaine effects on DAT-KD mice.
The DAT-KO and DAT-CI mice were generated using ES cells from 129Sv/J mice and crossed with C57BL6 mice, thus they have a mixed genetic background [17]. Several studies used mice that were backcrossed with C57BL6 mice for many generations [17]. The original DAT-KD mice were generated in 129Sv/J background but this strain does not respond well in many tests that are used to evaluate psychostimulant effects. Therefore, the DAT-KD mice used in this study were backcrossed to C57BL/6J for 8 to 9 generations and thus their background matches with the backgrounds of both the DAT-CI mice and DAT-KO mice. This is important because it has been shown that cocaine's effects are not the same in different strains of mice [21, 22]. In addition, DAT-KO mice backcrossed to different strain backgrounds also respond to cocaine differently [23].
In DAT-KD mice, DAT activity is reduced to 10% of the wild type level, resulting in a hyperdopaminergic tone and increased locomotor activity [18]. In addition, DAT-KD mice have an altered response to amphetamine, which might be due to an altered balance between DA autoreceptor and heteroreceptor functions [18]. Amphetamine elevates extracellular DA by interacting with DAT and, more importantly, with the vesicular monoamine transporter, inhibiting DA uptake, and inducing DA release from DA containing vesicles to the cytosol through the vesicular monoamine transporter and to the extracellular space through DAT [5]. In contrast, cocaine elevates extracellular DA by simply binding to DAT and inhibiting DA reuptake. Despite the vastly different mechanisms of action, amphetamine and cocaine both increase DA in the synapse and enhance dopaminergic neurotransmission, thereby stimulating locomotor activity and producing reward in animals. The fact that amphetamine produces locomotor inhibition instead of stimulation in DAT-KD mice suggested the possibility that cocaine effects might also be altered in these mice.
Fig. 1 shows that DAT-KD mice had considerably higher baseline locomotor activity than WT mice before drug or saline injection and this difference was statistically significant (Fig. 1C). This result confirms our previous observation [18] and is consistent with the hyperdopaminergic tone in these mice [18]. Fig. 1 also shows that cocaine clearly increased locomotor activity in DAT-KD mice as well as in WT mice. While cocaine was capable of increasing locomotor activity in both genotypes, there were differences in each genotype's response. For instance, both 5 and 10 mg/kg cocaine produced stronger locomotor stimulation in DAT-KD mice than it did in wild type mice (Fig. 1D). The reason for this difference is not clear but could be due to presynaptic or postsynaptic adaptive changes, such as reduced D2 autoreceptor signaling, in response to the hyperdopaminergic tone in DAT-KD mice. It is also not clear why the amphetamine effect is altered in DAT-KD mice while the cocaine effect is maintained. Our results demonstrate that a dramatically reduced DAT activity does not lead to a loss or a reduction of the stimulating effect of cocaine. This is in contrast to observations of DAT-KO mice and DAT-CI mice. In heterozygous DAT-KO mice, DAT activity is reduced by 50% and cocaine stimulates locomotion as well as in WT mice; while in homozygous DAT-KO mice, DAT is absent and cocaine has no effect on locomotion [24]. In DAT-CI mice, the mutated DAT is over 50-fold less sensitive to cocaine inhibition but has lower uptake activity. Instead of stimulation, cocaine suppresses locomotor activity in DAT-CI mice [17]. The fact that cocaine retains these effects in both heterozygous DAT-KO mice and DAT-KD mice indicates that the reduction of DAT expression and thus DAT activity to 50% and 10% of wild type mice does not by itself lead to adaptive changes that abolish the stimulating effect of cocaine and in fact, 10% DAT activity seems to be sufficient for the preservation of many normal functions, such as some cocaine responses. Therefore, the locomotor suppression observed in DAT-CI mice is not due to reduced DA clearance or changes in DA homeostasis but is due to the lack of cocaine inhibition of DAT and to cocaine effects on other cocaine targets. Cocaine inhibition of DAT, SERT, and NET and the resulting enhancement of neurotransmission in all three systems may contribute to the locomotor effect in wild type mice. Cocaine inhibition of SERT and/or NET in the absence of DAT inhibition in DAT-CI mice leads to locomotor suppression. It is not clear whether cocaine inhibition of SERT or NET enhances or dampens locomotor stimulation in wild type mice where DAT is also inhibited. Further studies are being performed to assess the role of SERT and NET in locomotor suppression.
Cocaine and other addictive drugs are known to produce reward in humans as well as in mice and other animals. CPP tests are commonly used to measure the rewarding properties of drugs. In this behavioral test, drug administration to animals is repeatedly paired with a set of environmental cues. When allowed to explore freely, the animals spend more time in the environment paired with a drug that produces reward.
Previously we have shown that up to 20 mg/kg cocaine is not enough to inhibit the cocaine-insensitive mutant DAT and elevate extracellular DA in DAT-CI mice [17]. We have also shown that cocaine lost its ability to produce reward in DAT-CI mice while amphetamine still produces CPP in these mice. Therefore, we conclude that the reward pathway in DAT-CI mice is functional and the lack of cocaine-induced reward in DAT-CI mice is due to the inability of cocaine to block the modified DAT. However, DAT activity in DAT-CI mice is significantly lower than that in WT mice. The DA clearance rate in DAT-CI mice is between those of DAT-KD mice and heterozygous DAT-KO mice [17]. It remains a concern that lowered DAT activity might lead to neuroadaptations that may alter cocaine effects. Thus, we examined whether cocaine is still able to produce reward in DAT-KD mice that have only 10% of wild type DAT expression. Fig. 2 shows that cocaine still produced robust CPP in DAT-KD mice. In addition, cocaine produces reward in heterozygous DAT-KO mice [9–11]. These results demonstrate that the reduction of DAT activity and elevated dopaminergic tone does not abolish cocaine's rewarding effect in mice.
Several mouse models have been generated with alterations to the DAT gene, providing excellent tools to study the functions of DAT and the mechanism of cocaine effects: DAT-KD mice [18] and heterozygous DAT-KO mice [24] with DAT expression reduced to 10% and 50% of the wild type level respectively, DAT-CI mice [17] with a cocaine-insensitive DAT mutant that is functional but with reduced uptake activity, and homozygous DAT-KO mice [24] with DAT completely deleted. Heterozygous DAT-KO mice [24]and DAT-KD mice have significantly elevated DA tone but in these mice DAT is blocked by cocaine and the mice respond to cocaine similarly to wild type mice. The adaptive changes to the elevated dopaminergic tone in these mice are likely to be moderate. The changes do not abolish cocaine's ability to produce reward or to stimulate locomotor activity. Therefore, it is likely that the mechanism of cocaine action in these mice is similar to that in wild type mice.
In DAT-CI mice, DA uptake activity is reduced and the basal DA tone is also significantly elevated [17]. Since the extent of basal DA elevation in DAT-CI mice is between those in heterozygous DAT-KO and DAT-KD mice [17], it is reasonable to assume that the adaptive changes in DAT-CI mice are also moderate and do not interrupt the drug reward pathway. We have shown that amphetamine still produces reward in DAT-CI mice suggesting a functional reward pathway [17]. Although the elevated basal DA tone is due to reduced DAT activity in all mouse lines, the causes of the reduction are different; reduced expression of wild type DAT, in heterozygous DAT-KO and DAT-KD mice, versus normal expression of a mutant DAT with lower uptake activity in DAT-CI mice. Therefore, it is still possible that the mutated DAT in DAT-CI mice may cause unique adaptive changes, by an unknown mechanism, which could abolish cocaine reward and locomotor stimulation.
In contrast, in homozygous DAT-KO mice DAT is completely lacking. Intriguingly, the elimination of DAT, the consequent severe alteration of DA homeostasis and related pathways, and the tremendous adaptive changes still fail to abolish the rewarding effect of cocaine [24]. However, the mechanism of cocaine reward in DAT-KO mice is very different from that in wild type mice. For example, inhibition of SERT or NET with selective inhibitors elevates DA levels in the nucleus accumbens and produces reward in DAT-KO mice but not in wild type mice [24]. These results indicate that the complete deletion of DAT significantly alters the mechanism of cocaine effects. It seems that cocaine-induced DA elevation in the nucleus accumbens is still critical and the signaling from serotoninergic or noradrenergic systems to the dopaminergic system might be altered. The exact mechanism is not clear and currently under intensive investigation.