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Official websites use. Share sensitive information only on official, secure websites. This work is licensed under a Creative Commons Attribution 4. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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. 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. Amphetamines are known to enhance extracellular dopamine levels, but the underlying mechanisms are unclear. Utilising a new pH biosensor for synaptic vesicles, the authors show that amphetamines diminish vesicle pH gradients, disrupting dopamine packaging and leading to increased neurotransmitter release. Amphetamines' psychostimulant effects are generally thought to result from increased extracellular dopamine mediated by efflux of cytoplasmic dopamine through the dopamine transporter DAT 2. How amphetamines mobilize dopamine from vesicles to the cytoplasm for subsequent efflux is less clear. Whether amphetamines also act directly on VMAT to redistribute dopamine from vesicles into the cytoplasm has been debated, and numerous mechanisms have been proposed 6. Amphetamines interact with VMAT in vitro , leading some investigators to conclude that they act as non-substrate inhibitors that elevate cytoplasmic dopamine by simply blocking its accumulation into vesicles and thus making more available for efflux 7. Others have inferred that amphetamines are substrates of VMAT that drive carrier-mediated exchange of vesicular monoamines into the cytoplasm 8 , 9. Amphetamines are weak bases pK a 8. Evidence for these diverse mechanisms has come in large part from in vitro studies of isolated vesicles, cells and brain slices, but the actual relevance of these proposed mechanisms to amphetamines' in vivo actions has not been ascertained. Earlier work by Dwoskin and colleagues showed that tetrabenazine and lobeline analogues, which are inhibitors of the neuronal VMAT isoform, VMAT2, blocked methamphetamine's behavioural action in rodents 16 , This selective antagonism indicates that VMAT function is required for the acute actions of amphetamines to release dopamine from intraluminal stores. To elucidate how amphetamines act on synaptic vesicles to release dopamine into the cytoplasm, we developed an experimental system in Drosophila melanogaster using a functionally viable ex vivo brain preparation Actions of amphetamines were studied in this whole-brain preparation using small synthetic and genetically-encoded fluorescent reporters visualized by multiphoton microscopy. We used the second-generation fluorescent false neurotransmitter FFN ref. To examine the effects of amphetamines on vesicle pH, we expressed the synaptic vesicle pH biosensor, dVMAT-pHluorin 18 , in dopaminergic neurons, which allowed us to monitor real-time changes in synaptic vesicle pH in whole living brain. Both cocaine and amphetamines are expected to produce locomotor and self-administration behaviours through the common step of elevating extracellular dopamine levels Mean and s. For self-administration rates 0. Building on these findings in rodents, we established a novel experimental system in D. The genetic tractability of Drosophila permits targeted manipulation of gene expression to determine contributions of individual genes to amphetamine's actions and the neuronal pathways in which they function. Using a behavioural assay of amphetamine-induced hyperlocomotion 20 , 21 , we first examined whether the sole Drosophila VMAT isoform, dVMAT, is required for amphetamine to produce its behavioural effects in larvae. The amphetamine-induced increase in crawling velocity was approximately fivefold greater in WT than in dVMA T null mutants, demonstrating that VMAT is essential for amphetamine-induced hyperlocomotion. See Supplementary Fig. To identify the presynaptic neuronal populations that mediate dVMAT's contribution to amphetamine-induced locomotion, we restricted dVMAT expression to different monoaminergic populations. These larvae had a baseline crawling velocity 0. Thus, dVMAT expression specifically in dopamine neurons plays a critical role in mediating amphetamine-stimulated behaviour in flies, consistent with voluminous data that dopamine is the primary mediator of amphetamine's psychomotor stimulatory action in mammals 2 , To analyse the mechanisms of monoamine loading and release from synaptic vesicles, we used an ex vivo whole fly brain preparation to optically monitor monoaminergic vesicle content by multiphoton microscopy. Labelling was observed in the antennal lobes, suboesophageal ganglion and the protocerebrum Fig. Residual signal was primarily autofluorescence from tracheal structures triangle 74 as well as some variable staining outside the neuropil. String-like structures in e — g are autofluorescent trachea. We observed strong FFN labelling of TH Rescue brains in a dense field of dopamine nerve terminals belonging to the MB-MV1 neurons that project to the mushroom bodies and have recently been implicated in associative learning 26 , 27 Fig. FFN also labelled presynaptic dopamine terminals in the suboesophageal ganglion, antennal lobes, mushroom bodies Fig. Therefore, by using FFN to label TH Rescue brains, we can specifically image dopamine terminals to examine the neurochemical mechanisms that regulate the content of dopamine vesicles in response to both exocytic and non-exocytic releasing stimuli. To study the kinetics of stimulated release in presynaptic dopamine nerve terminals, we first measured exocytic vesicle release from FFNlabelled dopamine terminals in TH Rescue brains. The initial lag before destaining was mainly attributable to the time needed for the KCl to reach the imaging chamber and for solution exchange Supplementary Fig. The sparse punctate staining resistant to KCl-depolarization may be due to the heterogeneous release properties of monoaminergic nerve terminals, as reported recently in mouse brain slices Projected image stacks of the protocerebral neuropil before left and after center treatments and kinetics of fluorescence decay in MB-MV1 region right from representative experiments. Thus, FFN has permitted us to monitor for the first time the dynamics of neurotransmitter content within intact dopaminergic vesicles in an ex vivo whole-brain preparation. We hypothesized that KCl, chloroquine and amphetamine operate through distinct mechanisms, and explored these mechanisms with the recently developed in vivo pH sensor dVMAT-pHluorin 18 , as described below. We characterized a genetically encoded fluorescent reporter of intraluminal pH to examine changes in monoamine vesicle pH. Both electrical stimulation Supplementary Fig. Under basal conditions, the percentage of dVMAT-pHluorin on the cell surface in our ex vivo whole fly brain preparation We expressed the dVMAT-pHluorin biosensor in presynaptic dopamine nerve terminals to directly monitor amphetamine-induced pH changes in dopamine synaptic vesicles in a whole brain for the first time. This amphetamine-induced rise in vesicle pH was sustained during continuous application of the drug by bath superfusion Supplementary Fig. Comparison with the WT genetic background b shows the vast shift in amphetamine potency due to the absence of functional dDAT in dDAT fmn mutant brains. Drugs were sequentially applied to the brain preparation by bath superfusion. Specifically, we asked whether passive diffusion of amphetamines across the plasma membrane supplies sufficient intracellular amphetamine to produce vesicular alkalization or whether it must be imported by the concentrative transporter dDAT. Methamphetamine-induced vesicle alkalization was also blocked in the dDAT null mutant background Supplementary Fig. This is consistent with chloroquine's ability to lipophilically diffuse across membranes and also demonstrates that the synaptic vesicles in dDAT null brains are capable of intraluminal alkalization. Next, we used a pharmacological strategy to block dVMAT to determine whether its function at the vesicle membrane is necessary for amphetamine-induced vesicle alkalization. The experiments above clearly demonstrated that amphetamines cause vesicle alkalization in dopamine terminals in an ex vivo whole-brain preparation. At low micromolar concentrations of amphetamines, this alkalization requires concentrative transporters at both the plasma membrane DAT and the synaptic vesicle membrane VMAT. We therefore tested whether proven VMAT substrates also alkalize vesicles. Critically, the property shared by these diverse compounds is their ability to be transported by VMAT. This proposed antiport mechanism for substrate-induced vesicular alkalization is not dependent on vesicle exocytosis. An alternative explanation for dVMAT-pHluorin brightening could be that vesicle exocytosis shifts the sensor from the acidic vesicle lumen to the neutral extracellular milieu. Given the relatively fast kinetics of exocytosis, images were acquired at higher frequency to detect rapid vesicular pH changes. Brief pressure ejection of KCl led to a prolonged, intense pH change Fig. We also used the same genetic background of TeTxLC co-expression with dVMAT-pHluorin to construct a vesicle intraluminal pH calibration curve using a cocktail of ionophores across a broad pH range see Methods ; the TeTxLC was required to avoid the potential confound of vesicle exocytosis during calibration. All traces show single-plane fluorescence intensity measured at ms intervals, integrated over the MB-MV1 region, and normalized to initial values. Although there is consensus that amphetamines produce behavioural effects by raising extracellular dopamine levels, diverse and often contradictory inferences have been drawn to explain the molecular basis of this effect. In order to reveal amphetamine's actions at the synaptic vesicle level, we have combined a number of novel, complementary genetic, optical and pharmacological approaches to address these questions. These data are consistent with work by Dwoskin and colleagues who have developed inhibitors of methamphetamine behavioural actions using lobeline analogues. During their structure-driven pre-clinical studies, this group recognized that the drugs shared the common property of being VMAT blockers Selectivity for VMAT2 over DAT is especially important for discriminating between these transporters as sites of action for amphetamines, which act at both. If this were the case, then VMAT inhibitors would mimic or enhance the behavioural effects of amphetamines. We used a fly model system to elucidate the mechanism of amphetamines' actions at synaptic vesicles in vivo. We recently introduced FFNs to enable rapid imaging of monoamine storage and release dynamics in vertebrate brain slice 30 , To focus specifically on dopaminergic terminals, we used flies genetically engineered to express dVMAT only in TH-expressing dopamine neurons. Thus, this probe serves as a sensitive and selective surrogate marker for dopamine content in small synaptic and large dense core vesicles While similar kinetics of amphetamine-induced destaining of previous generation FFN molecules loaded into brain slices has been shown 51 , this is the first report of amphetamine-induced dopamine vesicle content release in whole brain. Since vesicular pH plays an important role in storage and release of vesicle contents, we used the recently developed vesicular pH biosensor, dVMAT-pHluorin, as a tool to elucidate amphetamines' effects on intraluminal pH within intact vesicles in our whole-brain preparation. This approach differs from earlier uses of pHluorin biosensors to monitor vesicle dynamics during exo- and endocytosis 31 , Lipophilic weak bases like chloroquine cause vesicle alkalization and readily redistribute vesicle contents as we showed in whole brain with FFN However, two lines of evidence demonstrate that, at relevant concentrations, amphetamines do not work by the same mechanisms as chloroquine. The requirement for functional transporters at both the plasma membrane and the vesicular membrane for amphetamine action challenges the common assumption that lipophilic diffusion alone can deliver enough amphetamine to vesicles to alkalize vesicles, even at high micromolar concentrations of the drug. Altogether, these data provide the most direct evidence to date that the amphetamines, like dopamine and FFN, are bona fide substrates of VMAT and not merely inhibitors. Measurements of synaptic vesicle pH are typically made in cells or in isolated vesicles, and to our knowledge this study represents the first attempt to measure the intraluminal pH of synaptic vesicles in a whole living brain. Ionic manipulations used for calibrating pH measurements can lead to exocytosis, confounding interpretation of the data. This value is substantially more acidic than reported by Sturman et al. Our data lead to a model for how pharmacologically relevant concentrations of amphetamines increase extracellular dopamine: 1 DAT imports and concentrates amphetamines in the cytoplasm. Vesicle deacidification alters the protonation state of luminal dopamine, which might be sufficient to increase its diffusion across the membrane. Whether VMAT itself might be a route of dopamine release from vesicles 11 , 65 requires further study. Our previous work demonstrated that phosphorylation of DAT is essential for dopamine efflux 66 and for locomotor behaviour induced by amphetamine but not for the actions of methylphenidate, a competitive non-substrate inhibitor of DAT 20 , These results are consistent with the inference from our rodent behavioural data that competitive inhibition at DAT by amphetamines is not sufficient to produce behavioural effects at the concentrations tested. Because amphetamines are also substrates of norepinephrine transporter and serotonin transporter, and VMAT is present in adrenergic and serotonergic neurons 6 , the tandem actions of plasma membrane transporters and VMAT are likely important for amphetamine-induced release of other monoamines as well. Furthermore, our results demonstrate the first application of a novel experimental system that can be used to develop important new insights into the physiology of intact monoaminergic vesicles. This allowed us to achieve improved probe expression in the resulting homozygous fly strain compared with the initial description of the probe Drug treatments. In some experiments, drugs were also applied by air pressure ejection 0. To test effects on vesicular pH, brains were incubated with 0. An isolated, ex vivo whole adult fly brain preparation was obtained by rapid removal and microdissection of the brain from decapitated flies as previously described A significant advantage of this preparation is that following removal of head cuticle and connective tissues, drugs are applied directly to brain tissue at known concentrations. This whole-brain preparation was imaged with continuous flow on an Ultima multiphoton laser scanning microscope Prairie Technologies Bruker Corp. Edwards and have been previously characterized by Adam et al. The filtres were rapidly washed twice with 1. Assays were performed with conditions in duplicate or triplicate for each sample. Data in each assay were normalized to the mean control maximal uptake level within the respective assay without background subtraction, and the data from three separate experiments were pooled and analysed using GraphPad Prism version 5. Immunolabeling was performed in PBS containing 0. For stimulated release experiments, third instar larvae were prepared in chilled calcium-free HL3. To prevent exocytic release of vesicles in response to ionophore treatment, these experiments were performed in flies co-expressing tetanus toxin light chain in the same dopamine terminals. To determine the fraction of dVMAT-pHluorin expressed on the cell surface, we used previously described methods 31 , Using the brief acid wash method, we found that the percentage of dVMAT-pHluorin on the cell surface under basal conditions was This experimental system affords facile manipulation of drug concentrations. The timing by which drug solutions equilibrated in the imaging chamber was determined by flowing an auto-fluorescent green dye dissolved in PBS buffer dilution under conditions identical to those experimentally used to deliver drugs to fly brains. Data acquisition was performed with Prairie View software version 4. The dynamic range of dVMAT-pHluorin fluorescence intensity was calculated as a ratio of peak fluorescence intensity to initial fluorescence in response to KCl stimulation. All data were graphed using GraphPad Prism. Two hundred flies , females:males of the respective genotype were placed in bottles filled with standard medium and permitted to lay eggs with experiments commencing on the fourth day of egg-laying. Food coloring was added to the yeast paste to ascertain whether the larvae fed on the provided paste. Each dish containing a set of 1—3 larvae was placed on a cool-operated, evenly illuminated fluorescent light box positioned underneath a video camera Dalsa PTM60, Teledyne, Dalsa, Waterloo, Ontario, Canada , which captured a high-contrast video image of larval profiles over a featureless background. We used the Multi-Worm Tracker and Choreography software packages open source availability to track and quantify larval movement. The coefficient of determination R 2 corresponded to fitting experimentally-derived FFN destaining curves to a one phase exponential decay preceded by a plateau phase; all curve fittings were plotted using GraphPad Prism. IC 50 values were computed using a nonlinear, least-squares regression analysis using GraphPad Prism. Affinities K i values were calculated using the Cheng—Prusoff equation How to cite this article: Freyberg, Z. Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain. Markiavelli, Stephen Rayport and Leslie Vosshall for helpful discussion and comments on the manuscript as well as to Maryann Carrigan and Patty Ballerstadt for administrative assistance J. We also gratefully acknowledge the contributions of Dawn French-Evans in conducting both catalepsy and locomotor activity studies in mice and of Eve Vagg for assistance with figures. This work was financially supported by K08 DA Z. Gerstner, Jr, Scholars Program Z. Sames , G. Mathers Charitable Foundation D. Kirschstein T32 ES C. Kirschstein GM A. Author contributions Z. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Nat Commun. Find articles by Zachary Freyberg. Find articles by Mark S Sonders. Find articles by Jenny I Aguilar. Find articles by Takato Hiranita. Find articles by Caline S Karam. Find articles by Jorge Flores. Find articles by Andrea B Pizzo. Find articles by Yuchao Zhang. Find articles by Zachary J Farino. Find articles by Audrey Chen. Find articles by Ciara A Martin. Find articles by Theresa A Kopajtic. Find articles by Hao Fei. Find articles by Gang Hu. Find articles by Yi-Ying Lin. Find articles by Eugene V Mosharov. Find articles by Brian D McCabe. Find articles by Robin Freyberg. Find articles by Kandatege Wimalasena. Find articles by Ling-Wei Hsin. Find articles by Dalibor Sames. Find articles by David E Krantz. Find articles by Jonathan L Katz. Find articles by David Sulzer. Find articles by Jonathan A Javitch. Received Feb 2; Accepted Jan 6; Collection date All Rights Reserved. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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