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The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H(+) antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

Multiple populations of aminergic neurons are affected in Parkinson's disease (PD), with serotonergic and noradrenergic loci responsible for some non-motor symptoms. Environmental toxins, such as the dithiocarbamate fungicide ziram, significantly increase the risk of developing PD and the attendant spectrum of both motor and non-motor symptoms. The mechanisms by which ziram and other environmental toxins increase the risk of PD, and the potential effects of these toxins on aminergic neurons, remain unclear. To determine the relative effects of ziram on the synaptic function of aminergic versus non-aminergic neurons, we used live-imaging at the Drosophila melanogaster larval neuromuscular junction (NMJ). In contrast to nearly all other studies of this model synapse, we imaged presynaptic function at both glutamatergic Type Ib and aminergic Type II boutons, the latter responsible for storage and release of octopamine, the invertebrate equivalent of noradrenalin. To quantify the kinetics of exo- and endo-cytosis, we employed an acid-sensitive form of GFP fused to the Drosophila vesicular monoamine transporter (DVMAT-pHluorin). Additional genetic probes were used to visualize intracellular calcium flux (GCaMP) and voltage changes (ArcLight). We find that at glutamatergic Type Ib terminals, exposure to ziram increases exocytosis and inhibits endocytosis. By contrast, at octopaminergic Type II terminals, ziram has no detectable effect on exocytosis and dramatically inhibits endocytosis. In contrast to other reports on the neuronal effects of ziram, these effects do not appear to result from perturbation of the Ubiquitin Proteasome System (UPS) or calcium homeostasis. Unexpectedly, ziram also caused spontaneous and synchronized bursts of calcium influx (measured by GCaMP) and electrical activity (measured by ArcLight) at aminergic Type II, but not glutamatergic Type Ib, nerve terminals. These events are sensitive to both tetrodotoxin and cadmium chloride, and thus appear to represent spontaneous depolarizations followed by calcium influx into Type II terminals. We speculate that the differential effects of ziram on Type II versus Type Ib terminals may be relevant to the specific sensitivity of aminergic neurons in PD, and suggest that changes in neuronal excitability could contribute to the increased risk for PD caused by exposure to ziram. We also suggest that the fly NMJ will be useful to explore the synaptic effects of other pesticides associated with an increased risk of PD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.

Dopamine is cytotoxic and may play a role in the development of Parkinson's disease. However, its interaction with environmental risk factors such as pesticides remains poorly understood. The vesicular monoamine transporter (VMAT) regulates intracellular dopamine content, and we have tested the neuroprotective effects of VMAT in vivo using the model organism Drosophila melanogaster. We find that Drosophila VMAT (dVMAT) mutants contain fewer dopaminergic neurons than wild type, consistent with a developmental effect, and that dopaminergic cell loss in the mutant is exacerbated by the pesticides rotenone and paraquat. Overexpression of DVMAT protein does not increase the survival of animals exposed to rotenone, but blocks the loss of dopaminergic neurons caused by this pesticide. These results are the first to demonstrate an interaction between a VMAT and pesticides in vivo, and provide an important model to investigate the mechanisms by which pesticides and cellular DA may interact to kill dopaminergic cells., ((c) 2010 Elsevier Inc. All rights reserved.)

The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DA(cyt)) in cultured midbrain neurons, we confirm that elevated DA(cyt) and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DA(cyt) provide neuroprotection. L-DOPA increased DA(cyt) in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DA(cyt) was not altered by alpha-synuclein deletion, although dopaminergic neurons lacking alpha-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DA(cyt), and alpha-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets.

Mutations in human parkin have been identified in familial Parkinson's disease and in some sporadic cases. Here, we report that expression of mutant but not wild-type human parkin in Drosophila causes age-dependent, selective degeneration of dopaminergic (DA) neurons accompanied by a progressive motor impairment. Overexpression or knockdown of the Drosophila vesicular monoamine transporter, which regulates cytosolic DA homeostasis, partially rescues or exacerbates, respectively, the degenerative phenotypes caused by mutant human parkin. These results support a model in which the vulnerability of DA neurons to parkin-induced neurotoxicity results from the interaction of mutant parkin with cytoplasmic dopamine.

Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.

Drosophila melanogaster express vesicular transporters for the storage of neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation.

The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.

While a number of genome-wide association studies have identified microtubule-associated protein tau as a strong risk factor for Parkinson's disease (PD), little is known about the mechanism through which human tau can predispose an individual to this disease. Here, we demonstrate that expression of human wild-type tau is sufficient to disrupt the survival of dopaminergic neurons in a Drosophila model. Tau triggers a synaptic pathology visualized by vesicular monoamine transporter-pHGFP that precedes both the age-dependent formation of tau-containing neurofibrillary tangle-like pathology and the progressive loss of DA neurons, thereby recapitulating the pathological hallmarks of PD. Flies overexpressing tau also exhibit progressive impairments of both motor and learning behaviors. Surprisingly, contrary to common belief that hyperphosphorylated tau could aggravate toxicity, DA neuron degeneration is alleviated by expressing the modified, hyperphosphorylated tau(E14). Together, these results show that impairment of VMAT-containing synaptic vesicle, released to synapses before overt tauopathy may be the underlying mechanism of tau-associated PD and suggest that correction or prevention of this deficit may be appropriate targets for early therapeutic intervention.

Abstract: The exocytotic release of neurotransmitters requires active transport into synaptic vesicles and other types of secretory vesicles. Members of the SLC18 family perform this function for acetylcholine (SLC18A3, the vesicular acetylcholine transporter or VAChT) and monoamines such as dopamine and serotonin (SLC18A1 and 2, the vesicular monoamine transporters VMAT1 and 2, respectively). To date, no specific diseases have been attributed to a mutation in an SLC18 family member; however, polymorphisms in SLC18A1 and SLC18A2 may confer risk for some neuropsychiatric disorders. Additional members of this family include SLC18A4, expressed in insects, and SLC18B1, the function of which is not known. SLC18 is part of the Drug:H+ Antiporter-1 Family (DHA1, TCID 2.A.1.2) within the Major Facilitator Superfamily (MFS, TCID 2.A.1). [Copyright &y& Elsevier]

Mutations in human parkin have been identified in familial Parkinson's disease and in some sporadic cases. Here, we report that expression of mutant but not wild-type human parkin in Drosophila causes age-dependent, selective degeneration of dopaminergic (DA) neurons accompanied by a progressive motor impairment. Overexpression or knockdown of the Drosophila vesicular monoamine transporter, which regulates cytosolic DA homeostasis, partially rescues or exacerbates, respectively, the degenerative phenotypes caused by mutanthumanparkin. These results support a model in which the vulnerability ofDAneurons to parkin-induced neurotoxicity results from the interaction of mutant parkin with cytoplasmic dopamine. [ABSTRACT FROM AUTHOR]

The neurodegenerative effects of Parkinson's disease (PD) are marked by a selective loss of dopaminergic (DA) neurons. Epidemiological studies suggest that chronic exposure to the pesticide paraquat may increase the risk for PD and DA cell loss. However, combined exposure with additional fungicide(s) including maneb and/or ziram may be required for pathogenesis. To explore potential pathogenic mechanisms, we have developed a Drosophila model of chronic paraquat exposure. We find that while chronic paraquat exposure alone decreased organismal survival and motor function, combined chronic exposure to both paraquat and maneb was required for DA cell death in the fly. To initiate mechanistic studies of this interaction, we used additional genetic reagents to target the ubiquitin proteasome system, which has been implicated in some rare familial forms of PD and the toxic effects of ziram. Genetic inhibition of E1 ubiquitin ligase, but not the proteasome itself, increased DA cell death in combination with maneb but not paraquat. These studies establish a model for long-term exposure to multiple pesticides, and support the idea that pesticide interactions relevant to PD may involve inhibition of protein ubiquitination. [ABSTRACT FROM AUTHOR]

Physiologic and pathogenic changes in amine release induce dramatic behavioral changes, but the underlying cellular mechanisms remain unclear. To investigate these adaptive processes, we have characterized mutations in the Drosophila vesicular monoamine transporter (dVMAT), which is required for the vesicular storage of dopamine, serotonin, and octopamine. dVMAT mutant larvae show reduced locomotion and decreased electrical activity in motoneurons innervating the neuromuscular junction (NMJ) implicating central amines in the regulation of these activities. A parallel increase in evoked glutamate release by the motoneuron is consistent with a homeostatic adaptation at the NMJ. Despite the importance of aminergic signaling for regulating locomotion and other behaviors, adult dVMAT homozygous null mutants survive under conditions of low population density, thus allowing a phenotypic characterization of adult behavior. Homozygous mutant females are sterile and show defects in both egg retention and development; males also show reduced fertility. Homozygotes show an increased attraction to light but are mildly impaired in geotaxis and escape behaviors. In contrast, heterozygous mutants show an exaggerated escape response. Both hetero- and homozygous mutants demonstrate an altered behavioral response to cocaine. dVMAT mutants define potentially adaptive responses to reduced or eliminated aminergic signaling and will be useful to identify the underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster express vesicular transporters for the storage of neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation. • Drosophila melanogaster express orthologs of mammalian vesicular transporters. • Drosophila also express some vesicular transporters not found in mammals. • Mutants and transgenes available in Drosophila can be used to study transporter function. [ABSTRACT FROM AUTHOR]