Fly strains

D. melanogaster strains include the WT strain, w1118CS 10 (wCS10), which is w1118 outcrossed into Canton-S for 10 generations68. The dDAT null fumin mutant dDATfmn (gift of K. Kume, Kumamoto University)38, dVMAT null dVMAT1P169 and UAS-dVMAT transgene corresponding to the neuron-specific dVMAT-A isoform have been previously described38,68,69. GAL4 expression driver lines include TH-GAL4 (a gift of Dr S. Birman, Université Aix-Marseille II-III)22 and Tdc2-GAL424 which were previously shown to drive expression in DA and TA/OA neurons, respectively22,24. To detect pH changes in the intravesicular lumen of monoaminergic synaptic vesicles, we constructed a new fly strain with the novel transgene, UAS-dVMAT-pHluorin in both WT dDAT and dDATfmn genetic backgrounds. All fly strains were grown and maintained on standard cornmeal-molasses media at 25 °C under a 12-h light–dark schedule.

Construction of transgenic fly strains

The dVMAT-pHluorin fly strain was created by injection of the dVMAT-pHluorin probe sequence into WT fly embryos. The probe was generated by insertion of the pH-sensitive, super-ecliptic pHluorin DNA into the first luminal loop of dVMAT-A and surrounded by 5′ linker YPYDVPGSTSGGSGGTGG and 3′ linker SGGTGGSGGTGGSGGTGYAT between Arg-182 and Pro-183 of dVMAT-A. dVMAT-pHluorin was subsequently genetically recombined with the TH-GAL4 expression driver on chromosome III. This allowed us to achieve improved probe expression in the resulting homozygous fly strain compared with the initial description of the probe18.

dVMAT rescue fly strains were constructed by introducing the UAS-dVMAT transgene into the dVMATP1 null genetic background to selectively rescue dVMAT function in DA and OA/TA neurons using the TH-GAL4 and Tdc2-GAL4 expression drivers, respectively. The following genotypes were described in the text: (1) ‘TH Rescue’: w−; dVMATP1; TH-GAL4, UAS-dVMAT and (2) ‘Tdc Rescue’: w−; dVMATP1, Tdc2-GAL4; UAS-dVMAT. To examine dVMAT-pHluorin’s vesicular localization, we expressed the UAS-dVMAT-pHluorin transgene driven by the elav-GAL4 expression driver: elav-GAL4;;UAS-dVMAT-pHluorin. To test the effects of the respective expression drivers alone in the dVMAT null background, we used the following fly strains: (1) w−; dVMATP1; TH-GAL4 and (2) w−; dVMATP1, Tdc2-GAL4. Fly strains including the UAS-dVMAT transgene and dVMATP1 allele were also outcrossed for 10 generations into the wCS10 WT genetic background. To test whether dVMAT-pHluorin itself is functional in vivo, we expressed the UAS-dVMAT-pHluorin transgene driven by the Tdc2-GAL4 expression driver in the dVMAT null background using the w−; dVMATP1, Tdc2-GAL4; UAS-dVMAT-pHluorin fly strain, eliminating the potential confound of endogenous dVMAT expression.

Fly brain imaging

Drug treatments. All drugs were diluted in adult haemolymph-like saline (AHL; 108 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 8.2 mM MgCl 2 , 1 mM NaH 2 PO 4 , 10 mM sucrose, 5 mM trehalose, 5 mM HEPES, 4 mM NaHCO 3 ; pH 7.5, 265 mOsm). In most experiments, drugs were applied by bath superfusion into a flow chamber at room temperature (25 °C). Fluorescence was typically measured before treatment and after a 10 min drug equilibration period (25 °C). In some experiments, drugs were also applied by air pressure ejection (0.1–10 s) duration onto brains submerged in AHL as described earlier16. Drug was delivered to a pulled glass pipet directed at the MB-MV1 region at a distance of ∼10 μm away from the brain using Picospritzer II (Parker Hannifin Corporation, Cleveland, OH). To examine FFN206-labelling of presynaptic DA nerve terminals, we continuously superfused FFN206 (300 nM, 30 min) and examined brain neuropil by multiphoton laser scanning microscopy. To induce synaptic vesicle exocytosis or alkalization, we treated brains with KCl (40 mM, in AHL adjusted for ionic osmolarity) or CQ (100 μM) solutions, respectively. In experiments measuring destaining of FFN206 from TH Rescue brains, the brains were loaded to steady state with 300 nM FFN206 and then treated with drug solutions also containing 300 nM FFN206. To inhibit dVMAT activity, we pretreated brains with either reserpine or (+)-CYY477 (1 μM, 10–20 min) and imaged in the continuous presence of the respective blocker. To test effects on vesicular pH, brains were incubated with 0.1–700 μM amphetamine, methamphetamine, MPH, DA, FFN206, MPP+ or 3′-OHMPP+ in fly brains expressing dVMAT-pHluorin in presynaptic DA neurons in either a WT dDAT or dDATfmn genetic background. In experiments using DA, we pretreated brains with monoamine oxidase (MAO) inhibitors pargyline and selegiline (10 μM, 10 min).

Imaging. An isolated, ex vivo whole adult fly brain preparation was obtained by rapid removal and microdissection of the brain from decapitated flies as previously described16. 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., Middleton, WI). Fluorescent emission was collected using a 460 nm/50 nm FWHM bandpass emission filter for FFN206 and 525/50 nm FWHM bandpass filter for dVMAT-pHluorin.

Compounds

The drugs used in the present study including their respective salt and enantiomeric forms were as follows and purchased from Sigma-Aldrich (St Louis, MO) unless indicated otherwise: D-amphetamine hemisulphate, D-methamphetamine HCl, cocaine HCl (Merck, Whitehouse Station, NJ), methylphenidate HCl (MPH), bafilomycin A1 (Santa Cruz Biotechnology, Dallas, TX), 3-hydroxytyramine HCl (dopamine), 2-(N-morpholino)ethanesulfonic acid (MES), 1-methyl-4-phenylpyridinium iodide (MPP+), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), monensin A sodium salt (monensin), nigericin sodium salt (nigericin), N-methyl-N-propargylbenzylamine hydrochloride (pargyline HCl), reserpine, R(−)-N-α-dimethyl-N-2-propynyl-benzeneethanamine hydrochloride (selegiline HCl), N-Methyl-N-propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochloride (clorgyline HCl), 2-Cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide (entacapone), chloroquine diphosphate (CQ), (±)-tetrabenazine (TBZ), haloperidol, Ro4-1248, tetrodotoxin (Tocris Bioscience, Ellisville, MO), D-tubocurarine chloride (Tocris Bioscience), Triton X-100, digitonin (Santa Cruz Biotechnology), valinomycin, adenosine 5′-triphosphate magnesium salt (ATP), L-ascorbic acid, potassium sodium tartrate tetrahydrate, bovine serum albumin (fraction V; EMD Millipore, Billerica, MA), green food coloring dye (La Flor Products, Ridgewood, NY). [3H]dihydrotetrabenazine, [3H]nisoxetine, [3H]citalopram and [3H]dopamine were obtained from American Radiolabelled Chemicals (St Louis, MO), and [3H]WIN 35,428 was obtained from PerkinElmer Life Sciences (Waltham, MA). FFN206 (synthesized in the Department of Chemistry, Columbia University, New York, NY), 1-methyl-4-(3′-hydroxyphenyl)phenylpyridinium iodide (3′-OHMPP+; synthesized in the Dept. of Chemistry, Wichita State University, Wichita, KS) and (+)trans-10-desmethyltetrabenazine [(+)-CYY477] (synthesized at the School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan).

Preparation of (+)-CYY477

Preparation of (+)-CYY477 is described in detail in the Supplementary Information. Briefly, treatment of commercially available 7-hydroxy-6-methoxy-3,4-dihydroisoquinoline with 2-acetyl-N,N,N,4-tetramethylpentan-1-aminium iodide salt in refluxing ethanol provided racemic trans-10-desmethyltetrabenazine [(±)-CYY477] in 59% yield. Optical resolution of (±)-CYY477 using (+)-2,3-dibenzoyl tartaric acid ((+)-DBT) in ethanol yielded optically pure (+)-CYY477 (ee>99%) according to our modifications to previously described methods70 (see Supplementary Note 1, Supplementary Figs 10–13).

Assay of Dopamine Uptake by VMAT2

293-rV2 cells, HEK293 cells stably transfected with rat VMAT2, were obtained from Dr R.H. Edwards and have been previously characterized by Adam et al. (2008) and by Hu et al.17,71_ENREF_68. Cells were grown in DMEM in 10% fetal bovine serum, 100 units per ml penicillin, 100 μg ml−1 streptomycin (37 °C, 5% CO 2 ) in polystyrene plates to 85% confluency. On the day of assay, confluent cells were first washed with PBS, trypsinized, and washed in DMEM before being placed in Resuspension Buffer (110 mM L(+)-K-Na-tartaric acid, 5 mM MgCl 2 , 5 mM D-glucose, 20 mM HEPES-K, pH 7.4) at 37 °C. Cells were counted and resuspended at a concentration of ∼100,000 cells per 250 μl in Assay Buffer (Resuspension Buffer supplemented with 5 mM Mg-ATP, 1 mM ascorbate, 0.2% bovine serum albumin, 10 μM digitonin, 10 μM of the COMT inhibitor entacapone and 10 μM each of the MAO inhibitors selegiline, clorgyline, and pargyline). 250 μl aliquots of cells were preincubated (10 min, 37 °C) either with 25 μl of the respective test compound in Assay Buffer or with 25 μl of buffer without compound. Test compounds were dissolved in DMSO, which was always <0.1% final concentration in the assay; digitonin was dissolved in 100% ethanol (0.1% final concentration). Thereafter, 25 μl of [3H]dopamine (60 Ci mmol−1) suspended in Assay Buffer was added to the assay mixture at a final concentration of approximately 10 nM (300 μl final volume). After incubation with [3H]dopamine (10 min, 37 °C ), the assay mixture was diluted to 1.5 ml with ice cold Wash Buffer (85 mM NaCl, 85 mM KCl, 5 mM MgCl 2 , 20 mM HEPES-K, pH 7.4) and immediately vacuum-filtred with a Brandel 24 well manifold (Brandel Instruments) and captured on prewetted Whatman GF/B filters. The filtres were rapidly washed twice with 1.5 ml Wash Buffer and then dissolved in Safety Solve Complete Counting Cocktail liquid scintillant (Research Products International, Mt. Prospect, IL) and counted on a Packard Tri-Carb 2100 TR liquid scintillation analyzer (PerkinElmer, Hopkinton, MA) at an efficiency of ∼31%. Nonspecific uptake was defined using 10 μM Ro4-1248 and typically ranged from 1–2% of total accumulated counts (typically ∼10% of total counts added). 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 5.0, GraphPad Software, La Jolla, CA) with a four parameter logistic model.

dVMAT-pHluorin characterization

For both immunolabeling and live imaging experiments at the larval neuromuscular junction (segment A4, muscle 13), the UAS-dVMAT-pHluorin transgene was expressed in Type Ib nerve terminals using the elav-GAL4 expression driver. For immunolabeling, larval fillets were fixed in 4% paraformaldehyde. Immunolabeling was performed in PBS containing 0.1% Triton X 100 detergent and 5% normal goat serum using either a monoclonal antibody to GFP (1:500, Invitrogen Life Technologies, Carlsbad, CA) or rabbit antiserum to Drosophila synaptotagmin 1 (1:500 dilution, gift of N. Reist, Colorado State University) followed by the appropriate secondary antibodies (1:1,000 dilution) conjugated to either Alexa Fluor 488 or Alexa Fluor 555 (both from Jackson ImmunoResearch Laboratories, West Grove, PA), respectively. Images represent projected Z-series acquired via confocal imaging (1 μm/section). For stimulated release experiments, third instar larvae were prepared in chilled calcium-free HL3.1 saline (70 NaCl, 5 mM KCl, 4 mM MgCl 2 , 10 mM NaHCO 3 . 5 mM trehalose, 115 mM sucrose, 5 mM HEPES, adjusted to pH 7.3272) and recordings made in HL3.1 supplemented with 2 mM CaCl 2 and 7 mM L-glutamic acid (with the latter to inhibit muscle contractions). The stimulus (40 Hz, 10 V, 2 s) was applied using a suction electrode in contact with the free nerve root and stimulation-induced changes in dVMAT-pHluorin fluorescence were imaged at a rate of 20 images/s.

To characterize the pH response properties of dVMAT-pHluorin in DA nerve terminals of fly brain, we treated with 40 mM KCl to stimulate exocytic release of the biosensor across a range of extracellular pH’s (pH 5.5–8.3) using a modified version of AHL with equimolar substitution of KCl for NaCl. To estimate the intraluminal pH of intact dopaminergic synaptic vesicles in intact brain, we generated a pH calibration curve using dVMAT-pHluorin and a buffered cocktail of ionophores comprised of 20 μM FCCP, 10 μM valinomycin, 10 μM nigericin as well as 1 μM bafilomycin A1 and 0.1% Triton X-100(final concentrations)73 across a range of pH’s (pH 5.5–8.0) to permeabilize the vesicles and thus equilibrate the intraluminal vesicular pH with the respective extracellular pH. 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. We generated a pH calibration curve by graphing pH versus ΔF/F initial where F initial was obtained while superfusing AHL (pH 7.5) for 10 min without the ionophore cocktail. Points chosen for the calibration curve were 2 min following complete bath exchange with the respective cocktail and fitted with GraphPad Prism with a logistic model (Hill coefficient=1). For improved stability in these experiments, AHL was modified to include 9 mM HEPES (pH 6.5–8.3) or 9 mM MES (pH 5.0–6.0) and NaHCO 3 was excluded.

To determine the fraction of dVMAT-pHluorin expressed on the cell surface, we used previously described methods31,33. Specifically, we subjected whole, ex vivobrain preparations expressing dVMAT-pHluorin in presynaptic DA nerve terminals to a brief acid wash (100 s, 25 °C, pH 5.5) to quench fluorescence from cell surface-expressed biosensor, followed by recovery of fluorescence after wash-out (25 °C, pH 7.5). To confirm these values, we used complementary NH 4 Cl alkalization (50 mM, 25 °C, pH 7.5, 60 s) to visualize the intracellular vesicular dVMAT-pHluorin pool combined with KCl treatment (40 mM, 25 °C, pH 7.5, 60 s) to visualize both intracellular and externalized cell surface dVMAT-pHluorin fluorescence. Using the brief acid wash method, we found that the percentage of dVMAT-pHluorin on the cell surface under basal conditions was 14.5%±2.5%, comparable to 17.7%±5.4% determined with the complementary NH 4 Cl alkalization method.

Imaging

An isolated, ex vivo whole adult fly brain preparation was obtained by rapid removal and microdissection of the brain from decapitated flies as previously described16. This whole brain preparation was placed in a recording chamber (JG-23, Warner Instruments, Hamden, CT) with continuous flow of AHL. 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 (1:100 dilution) under conditions identical to those experimentally used to deliver drugs to fly brains. Brain preparations were imaged on an Ultima multiphoton laser scanning microscope (Prairie Technologies Bruker Corporation, Middleton, WI) using either a 63 × (0.9 numerical aperture (NA)) or a 20 × (1.0 NA) water immersion objective lens (Carl Zeiss Microscopy LLC, Thornwood, NY). The illumination source was a Coherent Chameleon Vision II Ti: Sapphire laser (Coherent, Inc., Santa Clara, CA) and we typically used <5 mW mean power at the sample. Fluorescent emission was collected using a 460 nm/50 nm FWHM bandpass emission filtre for FFN206 (λ ex =820 nm) and 525/50 nm FWHM bandpass filtre for dVMAT-pHluorin (λ ex =920 nm). When measuring the effect of FFN206 on dVMAT-pHluorin brightening, there was no cross-talk of the FFN206 signal into the dVMAT-pHluorin 525 nm/50 nm imaging channel when using λ ex =920 nm. We tested for spectral bleed-through and found that even a 1,000 μM solution of FFN206 generated no detectable fluorescence when using dVMAT-pHluorin excitation parameters (λ ex =920 nm, ∼1.5 mW mean power at sample). R3846 multi-alkali photomultiplier-tube detectors (Hamamatsu Photonics, Middlesex, NJ) were used for both FFN206 and dVMAT-pHluorin imaging. Data acquisition was performed with Prairie View software (version 4.0.29, Prairie Technologies Bruker Corporation, Middleton, WI).

Fixed larval fillet preparations were imaged on a Zeiss LSM5 Pascal Laser Scanning confocal microscope equipped with a Zeiss 63 × Neofluor, 1.3 NA oil immersion objective lens. Live imaging of these larval fillets was conducted using a Zeiss Axio Examiner Z1 microscope equipped with a cooled back-illuminated electron multiplying CCD camera (Andor iXon3 897, Andor, South Windsor, CT) and DG4 light source (Sutter Instrument, Novato, CA) with a GFP Brightline Filter Set (Semrock, Rochester, NY) and Zeiss Achroplan 100 × (1.0 NA) water-immersion objective lens.

Image processing and analysis

For experiments involving FFN206 or VMAT-pHluorin imaging in adult fly central brain, maximum-intensity z-projections were generated and quantified using the Fiji/ImageJ image processing package (National Institutes of Health, Bethesda, MD). Unless indicated otherwise, we normalized changes in images’ fluorescence intensity by using a ratio of fluorescence intensity change (ΔF, calculated as F−F baseline ) relative to the maximal fluorescence intensity change (ΔF max , calculated as F−F max ) based on treatment with chloroquine (100 μM, 25 °C), ΔF/ΔF max . In calibrating dVMAT-pHluorin’s pH-dependent fluorescence intensity changes in adult central brain, we normalized the respective fluorescence values to a ΔF max derived from 40 mM KCl, pH 8.3. dVMAT-pHluorin fluorescence intensity changes in larval fillet preparations during electrical stimulation were expressed as ΔF/F, which was calculated as (F−F baseline )/F baseline where F baseline is the average of intensities 2 s before stimulus. Maximal ΔF/F in this preparation was calculated as (F peak −F baseline )/F baseline , where F peak is the average of the 10 frames (0.5 s) acquired immediately following cessation of stimulation.

For curve fittings, images were corrected for background fluorescence and subsequently normalized to initial predrug treatment MB-MV1 fluorescence (F i ) using a custom program written in MATLAB (version R2012b, Mathworks, Natick, MA) and described in further detail elsewhere (Aguilar et al., in preparation). The decay time constant (τ decay ) and decay half-time (t 1/2 ) for FFN206 destaining were estimated using the least-squares method to fit fluorescence values to a single-exponential function preceded by a plateau phase: F/F i (t)=(F i -Plateau) e(−(t−t 0 )/τdecay)+Plateau where t 1/2 =τ decay ln2 and where F/F i (t) is the normalized fluorescence value as a function of time. Similarly, the time constant for decay of dVMAT-pHluorin fluorescence following electrical stimulation was estimated by fitting ΔF/F values to a single exponential function (using GraphPad Prism). The time constant (Tau, or 1/k) was calculated using the equation Y=(Y 0 −Y p )e−kt where Y 0 represents the peak after the stimulus and Y p represents the asymptotic, plateau value of Y at t=∞ with k as the rate constant of decay. The decay was represented as t 1/2 as calculated from the derived curve.

The signal to noise ratio (SNR) for FFN206 fluorescent signal was calculated as a ratio of the average FFN206 fluorescence in the MB-MV1 region to the standard deviation of background fluorescence. The dVMAT-pHluorin SNR was calculated as the ratio of the CQ-induced peak fluorescence to the s.d. of baseline fluorescence pre-drug treatment in MB-MV1. 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.

Larval locomotion assay

Two hundred flies (3:2, 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. twenty to 30 early third instar larvae (82- to 86-h old) per experimental group were washed with distilled water and placed onto 70% yeast paste (vehicle treatment) or 70% yeast paste in 60 mM amphetamine (Sigma, St Louis, MO) for 1 h (18 °C). Food coloring was added to the yeast paste to ascertain whether the larvae fed on the provided paste. Fed larvae were subsequently transferred onto 100-mm Petri dishes filled with 1% agar dissolved in distilled water. 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 PT-41-04M60, Teledyne, Dalsa, Waterloo, Ontario, Canada), which captured a high-contrast video image of larval profiles over a featureless background. Larvae were acclimated on the agar plate for 1 min followed by 1 min of data acquisition in a designated behaviour room (23–25 °C, 35–40% humidity). We used the Multi-Worm Tracker and Choreography software packages (open source availability) to track and quantify larval movement.

Rodent behaviour

See Supplementary Methods

Statistical analyses