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correspondence: Michael Freissmuth, Medical University of Vienna, Institute of Pharmacology, Center of Physiology and Pharmacology, Waehringer Straße 13A, A-1090 Vienna, Austria; Tel: +43-1-40160 31371; Fax: +43-1-40160 931300.
Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Austria
The level of dopamine transporters (DATs) in the neuronal plasma membrane shapes learning and motor coordination in mice. Mechanisms underlying the regulated internalization of DAT and its return to the cell surface have been intensively studied in heterologous cells and in neuronal cell bodies. However, whether this cycling also happens in synaptic boutons, or axon terminals, thought to be the major functional site for DAT expression, was an open question that Kearney and colleagues recently addressed in the JBC. They showed that DAT cycling in the presynaptic specialization of dopaminergic neurons is subject to control by a cell-autonomous loop comprising dopamine autoreceptors and metabotropic glutamate receptors. These results should inform future studies in neural development and motor learning.
Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGlu5, drove rapid biphasic DAT membrane trafficking in ex vivo striatal slices, with region-specific differences between ventral and dorsal striata.
Dopamine is required for motor coordination and for gauging saliency of external stimuli (reward or aversion). In a simplified representation, the dorsal (caudate nucleus and putamen) and the ventral striatum (nucleus accumbens and tubular striatum) control movement and reward seeking and are modulated by dopamine, which originates from neurons in the pars compacta of the substantia nigra (SNc) and from the ventral tegmental area (VTA), respectively. The importance of dopamine for motor coordination is evident in Parkinson’s disease: the initiation (and termination) of voluntary movement is impaired due to the loss of dopaminergic neurons in the SNc. Released dopamine is retrieved into the presynaptic specialization by the dopamine transporter DAT (solute carrier-6A3/SLC6A3). DAT shapes dopaminergic transmission by binding to and retrieving dopamine, which clears the synapse. By operating in relay with the vesicular monoamine transporter v-MAT2 (SLC18A2), DAT also maintains vesicular dopamine stores. DAT is a popular target for both therapeutic (e.g., methylphenidate in attention-deficit hyperactivity disorder) and illicit drugs (cocaine, amphetamines) (
). However, with more than 100 and 200 published studies on trafficking and endocytosis, respectively, our knowledge of DAT cycling readily beats that of SERT (by a factor of 2 or better). In fact, the signaling pathways that initiate DAT redistribution to various endocytic compartments in transfected cell lines are known in considerable detail (
). However, the main business of DAT is not done in the neuronal soma, but rather in the axonal boutons, which are small and very distant from the soma and its endomembrane compartments. It is not yet clear if the same mechanisms operate in the axonal boutons as in the soma; RAB11 (a regulator of endosomal recycling), for instance, is lost in the axonal compartment as neurons mature (
). The authors resorted to cell surface biotinylation in striatal slices from mice harboring a tetracycline transactivator specifically expressed in midbrain dopaminergic neurons, which thus could be manipulated to express a synthetic gene (a M3-muscarinic receptor exclusively activated by clozapine-N-oxide), shRNAs, or CRE recombinase (to excise genes in appropriately crossed mouse strains). The striatal slices contain the dense arborizations and thus the boutons of dopaminergic neurons. The slices also contain the synaptic boutons of the cortical glutamatergic projections from the motor cortex. Kearney and colleagues first go through their version of “Hershey heaven”(“to have one experiment that works, and keep doing it all the time”, ref.
): that is, a long list of cell surface biotinylation experiments to recapitulate and expand earlier key observations obtained in transfected cells. They show that Gq-coupled receptors, i.e., both the clozapine-N-oxide-activated M3-receptor and the endogenous metabotropic glutamate receptor-5 (mGluR5), drive a rapid and transient increase in DAT surface levels by triggering dopamine release, which activates D2-autoreceptors and downstream protein kinase Cβ. DAT reinternalization is likely due to the activation of a yet-unspecified PKC isoform stimulated by mGluR5. Thus, the first achievements by these authors were to define a cell-autonomous loop and to sort out the sequence of events: upon Gq stimulation, regulated surface insertion of DAT precedes endocytosis. The increases in DAT surface levels are not overwhelming (about 1.5-fold increase); however, the authors’ second major achievement is to confirm by fast scan cyclic voltammetry that these increases in DAT surface levels do translate into changes in extracellular dopamine levels and dopamine uptake rates.
Skilled movement requires training, which establishes neuronal circuits: dopamine in the dorsal striatum is essential for creating and maintaining effective motor circuits. Accordingly, Kearney and colleagues examined the impact of the (apparently physiological) biphasic changes in DAT activity on motor learning by using a battery of tests, which interrogated motor coordination and motor learning in mice. Their third major achievement is to show that, upon elimination of mGluR5 in striatal neurons – and thus of mGluR5-driven increases in DAT surface levels – motor performance of mice does not improve over time, but this can be corrected by inhibiting DAT.
Kearney et al. also examined Gq-driven DAT cycling in the ventral striatum: at the time resolution afforded by cell surface biotinylation, DAT cycling was more rapid in the ventral striatum than in the dorsal striatum. There behavioral outputs, which can be readily linked to VTA-dependent activation of the ventral striatum, were not explored. Dopaminergic neurons are not all equal (
): DAT cycling may differ in these individual subsets. The most conspicuous question pertains to the subset which harbors glutamate-containing vesicles and which co-releases dopamine and glutamate (
). In adult animals, dopaminergic neurons, which express the vesicular glutamate transporter-2 (vGluT2, SLC17A6), are rare in the SNc (<5%) but represent a substantial fraction (15%) in the VTA (
). It would be interesting to know if and how co-released glutamate regulates DAT surface levels in these neurons.
The dopamine hypothesis of schizophrenia posits that a reduced dopaminergic tone in the prefrontal cortex (and enhanced dopaminergic transmission in the striatum) contributes to phenotype. Dopaminergic neurons, which project from the VTA to the prefrontal cortex, express only low levels of DAT. However, higher levels of DAT were found in membranes prepared from the prefrontal cortices of patients suffering from schizophrenia than in those from control patients (
). This difference may be due to the disease rather than pharmacotherapy: most antipsychotics block D2-receptors. According to the observations of Kearney et al., D2-receptor inhibition is predicted to reduce DAT levels, if DAT cycling in mesocortical and nigrostriatal neurons obeys the same rules. At the very least, the insights from Kearney and colleagues may inspire others to interrogate the fate of DAT in different axonal territories.
Fig. 1Endocytotic recycling of the dopamine transporter is regulated by a cell-autonomous loop. The scheme summarizes the work of Kearney et al. (
) and of earlier work (reviewed in 3) in a highly simplified way: under basal conditions, midbrain dopaminergic neurons fire tonically. Hence, their axonal boutons (shown in green) release dopamine, which stimulates dopamine D2-receptors (D2-R). The activated D2-R initiates as protein kinase Cβ-dependent signaling cascade, which recruits the dopamine transporter (DAT) from an endosomal compartment (i.e., the retromer) to the cell surface (schematic rendering, left). The dendritic spines of striatal medium-sized spiny neurons (shown in blue) receive input from glutamatergic projections (shown in red), which presumably originate from the motor cortex and activate postsynaptic ionotropic and metabotropic receptors (not shown). The released glutamate also activates the Gq-coupled type I metabotropic glutamate receptor-5 (mGluR5) on the axonal terminals of the dopaminergic neuron. This enhances dopamine release and hence the translocation of DAT to the cell surface (schematic rendering, top), which translates into increased dopamine uptake. It is somewhat surprising that this multistep process is faster than the activation of an (unidentified classical or novel) protein kinase C (PKC)-isoform, which drives endocytosis of DAT (schematic rendering, right) and thus returns the system to the basal state.
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