Differential regulation of somatodendritic and nerve terminal dopamine release by serotonergic innervation of substantia nigra

Nigrostriatal dopaminergic neurons release dopamine from dendrites in substantia nigra and axon terminals in striatum. The cellular mechanisms for somatodendritic and axonal dopamine release are similar, but somatodendritic and nerve terminal dopamine release may not always occur in parallel. The current studies used in vivo microdialysis to simultaneously measure changes in dendritic and nerve terminal dopamine efflux in substantia nigra and ipsilateral striatum respectively, following intranigral application of various drugs by reverse dialysis through the nigral probe. The serotonin releasers (+/-)-fenfluramine (100 micro m) and (+)-fenfluramine (100 micro m) significantly increased dendritic dopamine efflux without affecting extracellular dopamine in striatum. The non-selective serotonin receptor agonist 1-(m-chlorophenyl)-piperazine (100 micro m) elicited a similar pattern of dopamine release in substantia nigra and striatum. NMDA (33 micro m) produced an increase in nigral dopamine of a similar magnitude to mCPP or either fenfluramine drug. However, NMDA also induced a concurrent increase in striatal dopamine. The D2 agonist quinpirole (100 micro m) had a parallel inhibitory effect on dopamine release from dendritic and terminal sites as well. Taken together, these data suggest that serotonergic afferents to substantia nigra may evoke dendritic dopamine release through a mechanism that is uncoupled from the impulse-dependent control of nerve terminal dopamine release.     

High doses of amphetamine augment, rather than disrupt, exocytotic dopamine release in the dorsal and ventral striatum of the anesthetized rat

High doses of amphetamine (AMPH) are thought to disrupt normal patterns of action potential-dependent dopaminergic neurotransmission by depleting vesicular stores of dopamine (DA) and inducing robust non-exocytotic DA release or efflux via dopamine transporter (DAT) reversal. However, these cardinal AMPH actions have been difficult to establish definitively in vivo. Here, we use fast-scan cyclic voltammetry (FSCV) in the urethane-anesthetized rat to evaluate the effects of 10 and 20 mg/kg AMPH on vesicular DA release and DAT function in dorsal and ventral striata. An equivalent high dose of cocaine (40 mg/kg) was also examined for comparison to psychostimulants acting preferentially by DAT inhibition. Parameters describing exocytotic DA release and neuronal DA uptake were determined from dynamic DA signals evoked by mild electrical stimulation previously established to be reinforcing. High-sensitivity FSCV with nanomolar detection was used to monitor changes in the background voltammetric signal as an index of DA efflux. Both doses of AMPH and cocaine markedly elevated evoked DA levels over the entire 2-h time course in the dorsal and ventral striatum. These increases were mediated by augmented vesicular DA release and diminished DA uptake typically acting concurrently. AMPH, but not cocaine, induced a slow, DA-like rise in some baseline recordings. However, this effect was highly variable in amplitude and duration, modest, and generally not present at all. These data thus describe a mechanistically similar activation of action potential-dependent dopaminergic neurotransmission by AMPH and cocaine in vivo. Moreover, DA efflux appears to be a unique, but secondary, AMPH action.     

Protein kinase C regulation of dopamine transporter initiated by nicotinic receptor activation in slices of rat prefrontal cortex

We previously reported that activation of nicotinic receptors causes an enhancement in amphetamine-stimulated release of dopamine via its transporter from slices of prefrontal cortex, but no such enhancement of release from slices of nucleus accumbens or striatum. The nicotinic receptors mediating the enhancement most likely contain alpha4 and beta2 subunits based upon pharmacological characterization. In this study, we sought to characterize the second messenger systems associated with the nicotine-mediated response. Sodium channel involvement was confirmed by the observation that tetrodotoxin blocked nicotine-mediated enhancement, whereas veratridine or elevated K(+) mimicked the enhancement seen with nicotine. Inclusion of EGTA blocked nicotine-mediated enhancement, suggesting that, even though no exogenous Ca(2+) was added, endogenous stores were required for the enhancement. The enhancement by nicotine was also abolished by the L-type voltage-dependent calcium channel (VDCC) antagonist nitrendipine, but not by the N-type VDCC antagonist omega-conotoxin GVIA. Finally, inhibition of protein kinase C also abolished the nicotine-mediated enhancement of amphetamine-stimulated dopamine release, whereas inhibitors of Ca(2+)/calmodulin kinase II did not. These findings establish that nicotine can exert selective effects on dopamine transporter activity in prefrontal cortex, an area involved in cognition and learning.     

Substrates dissociate dopamine transporter oligomers

Substrate-induced endocytic trafficking of dopamine transporter (DAT) has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in human embryonic kidney cells transiently or stably expressing N-terminal tagged DAT constructs. Dopamine (100 μM) or amphetamine (2–10 μM) reduced Myc-DAT coimmunoprecipitated along with Flag-DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. Dopamine (10–1000 μM) and amphetamine (0.2–200 μM) reduced DAT oligomerization as assessed by cross-linking with copper sulfate phenanthroline or Cu²⁺. Inhibition of endocytosis by 10 μM phenylarsine oxide or 450 mM sucrose counteracted the effect of 10 μM DA or 2 μM amphetamine in reducing DAT cross-linking. In addition to overall similarities between the results with the two cross-linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross-linking process and the sulfhydryl-reactive properties of phenylarsine oxide, respectively. The present results are the first to indicate regulation of oligomerization of an solute carrier family 6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT.     

Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.     

Repeated amphetamine treatment induces neurite outgrowth and enhanced amphetamine-stimulated dopamine release in rat pheochromocytoma cells (PC12 cells) via a protein kinase C- and mitogen activated protein kinase-dependent mechanism

Repeated intermittent treatment with amphetamine (AMPH) induces both neurite outgrowth and enhanced AMPH-stimulated dopamine (DA) release in PC12 cells. We investigated the role of protein kinases in the induction of these AMPH-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (100 nm and 300 nm), Ro31-8220 (300 nm) and the MAP kinase kinase inhibitor, PD98059 (30 micro m) inhibited the ability of AMPH to elicit both neurite outgrowth and the enhanced AMPH-stimulated DA release. The direct-acting PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 250 nm) mimicked the ability of AMPH to elicit neurite outgrowth and enhanced DA release. On the contrary, a selective PKA inhibitor, 100 micro m Rp-8-Br-cAMPS, blocked only the development of AMPH-stimulated DA release but not the neurite outgrowth. Treatment of the cells with acute AMPH elicited an increase in the activity of PKC and MAP kinase but not PKA. These results demonstrated that AMPH-induced increases in MAP kinase and PKC are important for induction of both the enhancement in transporter-mediated DA release and neurite outgrowth but PKA was only required for the enhancement in AMPH-stimulated DA release. Therefore the mechanisms by which AMPH induces neurite outgrowth and the enhancement in AMPH-stimulated DA release can be differentiated.     

Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).     

Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter

The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn²⁺, but the nature of the Zn²⁺-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn²⁺ on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn²⁺ depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn²⁺ to the outward facing conformation of DAT, which allows for an allosteric activation of DAT, in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn²⁺ dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn²⁺ on DAT that emulated all current experimental observations and accounted for all previous (in part contradictory) findings. Importantly, the model predicts that the intracellular Na⁺ concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn²⁺. This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).     

Regionally and functionally distinct serotonin3 receptors control in vivo dopamine outflow in the rat nucleus accumbens

Central serotonin(3) (5-HT(3)) receptors control the mesoaccumbens dopamine (DA) pathway. This control is thought to be conditional and might involve regionally distinct subpopulations of 5-HT(3) receptors. Here, using in vivo microdialysis in rats, we assessed the relative contribution of nucleus accumbens (Nacc) 5-HT(3) receptors to the overall influence exerted by 5-HT(3) receptors on accumbal DA release induced by different drugs or treatments. In freely moving rats, pre-treatment with 5-HT(3) antagonists (0.1 mg/kg ondansetron and/or 0.03 mg/kg MDL 72222, s.c.) reduced DA efflux enhanced by morphine (1-10 mg/kg, s.c.) and haloperidol (0.01 mg/kg, s.c.), but not amphetamine (1-2.5 mg/kg, i.p.) or cocaine (10-20 mg/kg, i.p.), the latter two drugs do not trigger depolarization-stimulated DA exocytosis. Intra-Nacc administration of ondansetron (1 microm) in freely moving rats reduced the DA effects elicited by 10 mg/kg morphine, but not 1 mg/kg morphine or haloperidol. The 5-HT(1A) agonist 8-OH-DPAT (0.1 mg/kg, s.c.), known to decrease central 5-HT tone, reduced 10 but not 1 mg/kg morphine-stimulated DA outflow in freely moving rats. In halothane-anaesthetized rats, intra-Nacc ondansetron (1 microm) application reduced dorsal raphe nucleus electrical stimulation (20Hz)-induced DA outflow. Our results show that regionally distinct populations of 5-HT(3) receptors control the depolarization-dependent exocytosis of DA and suggest that the involvement of Nacc 5-HT(3) receptors occurs only when central DA and 5-HT tones are concomitantly increased.     

A genetic screen in Caenorhabditis elegans for dopamine neuron insensitivity to 6-hydroxydopamine identifies dopamine transporter mutants impacting transporter biosynthesis and trafficking

The presynaptic dopamine (DA) transporter (DAT) is a major determinant of synaptic DA inactivation, an important target for psychostimulants including cocaine and amphetamine, and a mediator of DA neuron vulnerability to the neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion. To exploit genetic approaches for the study of DATs and neural degeneration, we exploited the visibility of green fluorescent protein (GFP)-tagged DA neurons in transgenic nematodes to implement a forward genetic screen for suppressors of 6-OHDA sensitivity. In our initial effort, we identified three novel dat-1 alleles conferring 6-OHDA resistance. Two of the dat-1 alleles derive from point mutations in conserved glycine residues (G55, G90) in contiguous DAT-1 transmembrane domains (TM1 and TM2, respectively), whereas the third allele results in altered translation of the transporter's COOH terminus. Our studies reveal biosynthetic, trafficking and functional defects in the DAT-1 mutants, exhibited both in vitro and in vivo. These studies validate a forward genetic approach to the isolation of DA neuron-specific toxin suppressors and point to critical contributions of the mutated residues, as well as elements of the DAT-1 COOH terminus, to functional expression of catecholamine transporters in neurons.     

Evidence for D2 receptor mediation of amphetamine-induced normalization of locomotion and dopamine transporter function in hypoinsulinemic rats

Dopamine (DA) D2 receptors regulate DA transporter (DAT) activity, and mediate some behavioral effects of amphetamine. DA clearance and amphetamine-stimulated locomotion are reduced in hypoinsulinemic [streptozotocin (STZ)-treated] rats, and these deficits are normalized by repeated treatment with amphetamine. Here, a role for D2 receptors in mediating amphetamine-induced normalization of these parameters was investigated. One week after a saline or STZ injection (50 mg/kg), rats were treated with amphetamine (1.78 mg/kg), raclopride (0.056 mg/kg), saline, or combinations thereof, every-other-day for 8 days with locomotor activity measured following each treatment. Conditioned place preference (CPP) for amphetamine and in vivo chronoamperometry to measure DA clearance were carried out on days 17 and 18, respectively, after STZ or saline. Baseline locomotion and DA clearance were significantly reduced in STZ-treated rats compared with control rats. In STZ-treated rats, amphetamine treatment normalized DA clearance, and restored the locomotor-stimulating effects of amphetamine. Raclopride prevented normalization of these parameters. Amphetamine produced CPP in both STZ-treated and control rats; raclopride significantly attenuated amphetamine-induced CPP in control and not in STZ-treated rats. These results support a role for D2 receptors in regulating DA transporter activity, and further demonstrate that D2 receptors contribute to changes in sensitivity to amphetamine in hypoinsulinemic rats.     

Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice

The vesicular monoamine transporter 2 (VMAT2) plays a pivotal role in regulating the size of vesicular and cytosolic dopamine (DA) storage pools within the CNS, and can thus influence extracellular DA neurotransmission. Transgenic mice have been generated with a dramatically reduced (by approximately 95%) expression of the VMAT2 gene which, unlike complete knockout lines, survive into adulthood. We compared the pre-synaptic regulation of both impulse-dependent (exocytotic) and carrier-mediated (via reversal of the DA transporter, DAT) DA release in the dorsolateral caudate putamen (CPu) of striatal slices derived from adult homozygous VMAT2 mutant and wild-type mice using fast cyclic voltammetry. Impulse-dependent DA release, evoked by a single electrical pulse, was lower in homozygous (116 nm) than wild-type mice (351 nm) indicating smaller vesicular DA stores, an observation supported by the evanescent effect of amfonelic acid (300 nm) in homozygous mice. Amphetamine (2 microm) increased extracellular DA via DAT reversal in both wild-type (by 459 nm) and VMAT2 mutant (by 168 nm, p < 0.01 vs. wild-type) mice. In both cases, the effect was blocked by the DAT inhibitor GBR12935 (1 microm). Simultaneously, amphetamine decreased impulse-dependent DA release, albeit less in homozygous (by 55%) than in wild-type (by 78%) mice. In wild-types, this decrement was largely reversed by GBR12935 but not by the D2/D3 autoreceptor antagonist (-)sulpiride (1 microm). Conversely, in homozygous VMAT2 mutant mice, it was attenuated by (-)sulpiride but not GBR12935. The D2/D3 receptor agonist quinpirole inhibited impulse-dependent DA release with a lower EC50 value in homozygous mice (12 nm) compared with wild-types (34 nm), indicating the compensatory presence of functionally supersensitive release-regulating autoreceptors. However, analysis of DA reuptake kinetics obtained in the absence and presence of DAT blockade (by cocaine and amfonelic acid) revealed only minor differences in DAT functionality. These results demonstrate that impaired vesicular DA storage constrains extracellular DA levels in the dorsolateral CPu whether induced by either impulse-dependent or carrier-mediated mechanisms and that the relative importance of the DAT and terminal autoreceptors as control mechanisms in the actions of amphetamine are reversed in VMAT2 mutant mice.     

Deficits in dopamine clearance and locomotion in hypoinsulinemic rats unmask novel modulation of dopamine transporters by amphetamine

Insulin affects brain reward pathways and there is converging evidence that this occurs through insulin regulation of the dopamine (DA) transporter (DAT). In rats made hypoinsulinemic by fasting, synaptosomal DA uptake is reduced. Interestingly, [3H]DA uptake is increased in hypoinsulinemic rats with a history of amphetamine self-administration. The possibility that amphetamine and insulin act in concert to regulate DAT activity prompted this study. Here we show that [3H]DA uptake, measured in vitro and clearance of exogenously applied DA in vivo, is significantly reduced in rats made hypoinsulinemic by a single injection of streptozotocin. Strikingly, amphetamine (1.78 mg/kg, given every other day for 8 days) restored DA clearance in streptozotocin-treated rats but was without effect on DA clearance in saline-treated rats. Basal locomotor activity of streptozotocin-treated rats was lower compared to control rats; however, in streptozotocin-treated rats, hyperlocomotion induced by amphetamine increased over successive amphetamine injections. In saline-treated rats the locomotor stimulant effect of amphetamine remained stable across the four amphetamine injections. These results provide exciting new evidence that actions of amphetamine on DA neurotransmission are insulin-dependent and further suggest that exposure to amphetamine may cause long-lasting changes in DAT function.