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The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain-binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain-binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

Addiction to psychostimulants (ie, amphetamines and cocaine) imposes a major socioeconomic burden. Prevention and treatment represent unmet medical needs, which may be addressed, if the mechanisms underlying psychostimulant action are understood. Cocaine acts as a blocker at the transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), but amphetamines are substrates that do not only block the uptake of monoamines but also induce substrate efflux by promoting reverse transport. Reverse transport has been a focus of research for decades but its mechanistic basis still remains enigmatic. Recently, transporter-interacting proteins were found to regulate amphetamine-triggered reverse transport: calmodulin kinase IIα (αCaMKII) is a prominent example, because it binds the carboxyl terminus of DAT, phosphorylates its amino terminus, and supports amphetamine-induced substrate efflux in vitro. Here, we investigated whether, in vivo, the action of amphetamine was contingent on the presence of αCaMKII by recording the behavioral and neurochemical effects of amphetamine. Measurement of dopamine efflux in the dorsal striatum by microdialysis revealed that amphetamine induced less dopamine efflux in mice lacking αCaMKII. Consistent with this observation, the acute locomotor responses to amphetamine were also significantly blunted in αCaMKII-deficient mice. In addition, while the rewarding properties of amphetamine were preserved in αCaMKII-deficient mice, their behavioral sensitization to amphetamine was markedly reduced. Our findings demonstrate that amphetamine requires the presence of αCaMKII to elicit a full-fledged effect on DAT in vivo: αCaMKII does not only support acute amphetamine-induced dopamine efflux but is also important in shaping the chronic response to amphetamine.

Dysfunction of brain dopamine transporter (DAT) has been associated with sensation seeking and impulse-control disorders. We recently generated a new animal model by stereotaxical inoculation of lentiviral vectors, which allowed localized intra-accumbal delivery of modulators for DAT gene: GFP (green fluorescent protein) control, silencers (Sil), a regulatable enhancer (DAT+), or both (DAT+Sil). Wistar male rats were followed both for socio-emotional profiles and for propensity to seek risky, uncertain rewards. Elevated anxiety and affiliation towards an unfamiliar partner emerged in Sil rats. Interestingly, in DAT+Sil rats (and Sil rats to a lesser extent) levels of playful social interaction were markedly reduced compared to controls. These DAT+Sil rats displayed a marked ‘gambling-like’ profile (i.e. preference for a large/uncertain over a small/sure reward), which disappeared upon doxycycline-induced switch-off onto DAT enhancer, but consistently reappeared with doxycycline removal. MRI-guided ¹H-MRS (at 4.7 T) examinations in vivo (under anaesthesia) revealed changes in the bioenergetic metabolites (phosphocreatine and total creatine) for DAT+Sil rats, indicating a functional up-regulation of dorsal striatum (Str) and conversely a down-regulation of ventral striatum (i.e. nucleus accumbens, NAc). A combined profile of (1) enhanced proneness to gambling and (2) strong social withdrawal is thus associated with altered DAT-induced balance within forebrain dopamine systems. In fact, risk of developing a gambling-prone, social-avoidant psychopathology might be associated with (1) dominant semi-automatic strategies and/or habits, developed within Str circuits, and (2) reduced NAc function, with poorer feedback adjustment on decisions by aversive experiences.

Dysfunction of brain dopamine transporter (DAT) has been associated with sensation seeking and impulse-control disorders. We recently generated a new animal model by stereotaxical inoculation of lentiviral vectors, which allowed localized intra-accumbal delivery of modulators for DAT gene: GFP (green fluorescent protein) control, silencers (Sil), a regulatable enhancer (DAT+), or both (DAT+Sil). Wistar male rats were followed both for socio-emotional profiles and for propensity to seek risky, uncertain rewards. Elevated anxiety and affiliation towards an unfamiliar partner emerged in Sil rats. Interestingly, in DAT+Sil rats (and Sil rats to a lesser extent) levels of playful social interaction were markedly reduced compared to controls. These DAT+Sil rats displayed a marked ‘gambling-like’ profile (i.e. preference for a large/uncertain over a small/sure reward), which disappeared upon doxycycline-induced switch-off onto DAT enhancer, but consistently reappeared with doxycycline removal. MRI-guided ¹H-MRS (at 4.7 T) examinations in vivo (under anaesthesia) revealed changes in the bioenergetic metabolites (phosphocreatine and total creatine) for DAT+Sil rats, indicating a functional up-regulation of dorsal striatum (Str) and conversely a down-regulation of ventral striatum (i.e. nucleus accumbens, NAc). A combined profile of (1) enhanced proneness to gambling and (2) strong social withdrawal is thus associated with altered DAT-induced balance within forebrain dopamine systems. In fact, risk of developing a gambling-prone, social-avoidant psychopathology might be associated with (1) dominant semi-automatic strategies and/or habits, developed within Str circuits, and (2) reduced NAc function, with poorer feedback adjustment on decisions by aversive experiences.