Fluorescent dopamine tracer resolves individual ... · Fluorescent dopamine tracer resolves...

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Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain Pamela C. Rodriguez a , Daniela B. Pereira b , Anders Borgkvist b , Minerva Y. Wong b , Candace Barnard b , Mark S. Sonders b,c , Hui Zhang c , Dalibor Sames a,1 , and David Sulzer b,c,d,e,1 a Department of Chemistry, Columbia University, New York, NY 10027; Departments of b Neurology, c Psychiatry, and d Pharmacology, Columbia University Medical Center, New York, NY 10032; and e Department of Neuroscience, New York Psychiatric Institute, New York, NY 10032 Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved November 28, 2012 (received for review August 7, 2012) We recently introduced uorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater uorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the mea- surement of individual synaptic terminal activity by following uorescence loss upon stimulation. Thus, FFN102 enables not only the identication of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allow- ing for the study of both the microanatomy and functional plas- ticity of the normal and diseased nervous system. dopamine reporter | secretion kinetics | molecular design | multiphoton imaging D opamine neurotransmission plays a key role in habit learn- ing, motivation, reward, and motor function (1), and altered dopamine neurotransmission is associated with disorders such as Parkinsons disease, schizophrenia, and drug addiction (24). As a socialneurotransmitter that overows relatively long distances beyond its presynaptic terminals, dopamines extrasynaptic concen- tration is principally determined by the combination of exocytotic neurotransmitter release and reuptake by the plasma membrane dopamine transporter (DAT) (5). Psychostimulants, such as co- caine and amphetamine (AMPH), increase extracellular dopamine via interactions with DAT. Extracellular dopamine concentration, particularly in the striatum where it is present at high levels, has been characterized by microdialysis (6, 7) and rapid electrochemical detection using carbon ber cyclic voltammetry (8, 9) and amperometry (10). The excellent temporal resolution of the electrochemical meth- ods is well suited for measuring changes in extrasynaptic dopa- mine concentration associated with neuronal activity. However, these approaches usually measure the release and reuptake of dopamine from large sets of striatal dopamine release sites and lack the spatial resolution required to study synaptic trans- mission at the level of individual presynaptic terminals. Optical methods provide vastly improved spatial resolution so that processes by which specic synapses are modulated can be studied. The rst group of uorescent reporters for the study of presynaptic function were the endocytic FM dyes (11), which act as tracers of exocytosis and endocytosis. These labels, however, do not indicate transmitter accumulation or release, but rather the fusion of synaptic vesicle membrane with the plasma mem- brane. Moreover, the FM dyes label all active presynaptic ter- minals with recycling synaptic vesicles regardless of the identity of the neurotransmitter. Another approach uses genetically encoded vesicle membrane proteins known as synaptopHluorins (12, 13) that exhibit pH-sensitive uorescence emission, although these also indicate vesicle membrane fusion rather than neuro- transmitter uptake and release. As a different approach, we recently introduced uorescent false neurotransmitters (FFNs) as uorescent tracers of neuro- transmitter uptake and release from individual presynaptic ter- minals (14). In our efforts to develop a second generation of FFNs, we have focused on the design of highly selective, pH- responsive dopamine-mimicking probes (15). Here, we introduce FFN102 as a pH-responsive uorescent DAT substrate that exhibits high selectivity for dopamine neuronal cell bodies in the midbrain and presynaptic terminals in the dorsal striatum. In addition to the identication of dopamine cells and their pro- cesses in acute mouse brain tissue, FFN102 enables the optical measurement of important functional parameters including DAT activity and the modulation of dopamine release within large populations of striatal dopamine terminals. Results Photophysical Characterization of FFN102. FFN102 (Fig. 1A) was designed as a uorescent pH reporter responsive at pH values relevant to synaptic vesicle physiology (pH 57) (15). Phenols exhibit pH-dependent photophysical properties (light absorp- tion, and in some cases, excitation and emission spectra) due to the equilibrium between the protonated phenol and deproto- nated phenolate forms. The phenol group in the 7-position of the coumarin nucleus renders FFN102 ratiometric in the excitation mode with a pK a value of 6.2. The absorption spectra of FFN102 exhibited pH dependence: the absorption maximum shifts to- ward 331 nm at lower pH values and to 371 nm at higher pH values, corresponding to the protonated and deprotonated forms, respectively (Fig. 1B). As expected, the uorescence ex- citation of this probe was also pH dependent, showing a peak at 340 nm at pH 5.0, mimicking the relatively acidic vesicular pH, and a peak at 370 nm at pH 7.4, the approximate pH of the cytoplasm (Fig. 1C). Although the emission wavelength is in- dependent of pH with a maximum at 453 nm, the intensity of emission is highly pH dependent (Fig. 1D). Thus, pH can be measured ratiometrically via optical means using two different excitation wavelengths (15). Pharmacological Characterization of FFN102. FFN102 was originally identied as a vesicular monoamine transporter 2 (VMAT2) substrate using VMAT2-transfected HEK cells (15). The present imaging studies in mouse brain tissue demonstrate that FFN102 is Author contributions: P.C.R., D. Sames, and D. Sulzer designed research; P.C.R., D.B.P., A.B., M.Y.W., C.B., M.S.S., and H.Z. performed research; D. Sames contributed new re- agents/analytic tools; P.C.R., D.B.P., A.B., M.Y.W., M.S.S., H.Z., and D. Sulzer analyzed data; and P.C.R., D.B.P., A.B., D. Sames, and D. Sulzer wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1213569110/-/DCSupplemental. 870875 | PNAS | January 15, 2013 | vol. 110 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1213569110

Transcript of Fluorescent dopamine tracer resolves individual ... · Fluorescent dopamine tracer resolves...

Page 1: Fluorescent dopamine tracer resolves individual ... · Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain Pamela C. Rodrigueza,

Fluorescent dopamine tracer resolves individualdopaminergic synapses and their activity in the brainPamela C. Rodrigueza, Daniela B. Pereirab, Anders Borgkvistb, Minerva Y. Wongb, Candace Barnardb,Mark S. Sondersb,c, Hui Zhangc, Dalibor Samesa,1, and David Sulzerb,c,d,e,1

aDepartment of Chemistry, Columbia University, New York, NY 10027; Departments of bNeurology, cPsychiatry, and dPharmacology, Columbia UniversityMedical Center, New York, NY 10032; and eDepartment of Neuroscience, New York Psychiatric Institute, New York, NY 10032

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved November 28, 2012 (received for review August 7, 2012)

We recently introduced fluorescent false neurotransmitters (FFNs)as optical tracers that enable the visualization of neurotransmitterrelease at individual presynaptic terminals. Here, we describea pH-responsive FFN probe, FFN102, which as a polar dopaminetransporter substrate selectively labels dopamine cell bodies anddendrites in ventral midbrain and dopaminergic synaptic terminalsin dorsal striatum. FFN102 exhibits greater fluorescence emissionin neutral than acidic environments, and thus affords a means tooptically measure evoked release of synaptic vesicle content intothe extracellular space. Simultaneously, FFN102 allows the mea-surement of individual synaptic terminal activity by followingfluorescence loss upon stimulation. Thus, FFN102 enables not onlythe identification of dopamine cells and their processes in braintissue, but also the optical measurement of functional parametersincluding dopamine transporter activity and dopamine release atthe level of individual synapses. As such, the development ofFFN102 demonstrates that, by bringing together organic chemistryand neuroscience, molecular entities can be generated that matchthe endogenous transmitters in selectivity and distribution, allow-ing for the study of both the microanatomy and functional plas-ticity of the normal and diseased nervous system.

dopamine reporter | secretion kinetics | molecular design |multiphoton imaging

Dopamine neurotransmission plays a key role in habit learn-ing, motivation, reward, and motor function (1), and altered

dopamine neurotransmission is associated with disorders such asParkinson’s disease, schizophrenia, and drug addiction (2–4). As a“social” neurotransmitter that overflows relatively long distancesbeyond its presynaptic terminals, dopamine’s extrasynaptic concen-tration is principally determined by the combination of exocytoticneurotransmitter release and reuptake by the plasma membranedopamine transporter (DAT) (5). Psychostimulants, such as co-caine and amphetamine (AMPH), increase extracellular dopaminevia interactions with DAT.Extracellular dopamine concentration, particularly in the

striatum where it is present at high levels, has been characterizedby microdialysis (6, 7) and rapid electrochemical detection usingcarbon fiber cyclic voltammetry (8, 9) and amperometry (10).The excellent temporal resolution of the electrochemical meth-ods is well suited for measuring changes in extrasynaptic dopa-mine concentration associated with neuronal activity. However,these approaches usually measure the release and reuptake ofdopamine from large sets of striatal dopamine release sites andlack the spatial resolution required to study synaptic trans-mission at the level of individual presynaptic terminals.Optical methods provide vastly improved spatial resolution so

that processes by which specific synapses are modulated can bestudied. The first group of fluorescent reporters for the study ofpresynaptic function were the endocytic FM dyes (11), which actas tracers of exocytosis and endocytosis. These labels, however,do not indicate transmitter accumulation or release, but ratherthe fusion of synaptic vesicle membrane with the plasma mem-brane. Moreover, the FM dyes label all active presynaptic ter-minals with recycling synaptic vesicles regardless of the identityof the neurotransmitter. Another approach uses genetically

encoded vesicle membrane proteins known as synaptopHluorins(12, 13) that exhibit pH-sensitive fluorescence emission, althoughthese also indicate vesicle membrane fusion rather than neuro-transmitter uptake and release.As a different approach, we recently introduced fluorescent

false neurotransmitters (FFNs) as fluorescent tracers of neuro-transmitter uptake and release from individual presynaptic ter-minals (14). In our efforts to develop a second generation ofFFNs, we have focused on the design of highly selective, pH-responsive dopamine-mimicking probes (15). Here, we introduceFFN102 as a pH-responsive fluorescent DAT substrate thatexhibits high selectivity for dopamine neuronal cell bodies in themidbrain and presynaptic terminals in the dorsal striatum. Inaddition to the identification of dopamine cells and their pro-cesses in acute mouse brain tissue, FFN102 enables the opticalmeasurement of important functional parameters includingDAT activity and the modulation of dopamine release withinlarge populations of striatal dopamine terminals.

ResultsPhotophysical Characterization of FFN102. FFN102 (Fig. 1A) wasdesigned as a fluorescent pH reporter responsive at pH valuesrelevant to synaptic vesicle physiology (pH 5–7) (15). Phenolsexhibit pH-dependent photophysical properties (light absorp-tion, and in some cases, excitation and emission spectra) due tothe equilibrium between the protonated phenol and deproto-nated phenolate forms. The phenol group in the 7-position of thecoumarin nucleus renders FFN102 ratiometric in the excitationmode with a pKa value of 6.2. The absorption spectra of FFN102exhibited pH dependence: the absorption maximum shifts to-ward 331 nm at lower pH values and to 371 nm at higher pHvalues, corresponding to the protonated and deprotonatedforms, respectively (Fig. 1B). As expected, the fluorescence ex-citation of this probe was also pH dependent, showing a peak at340 nm at pH 5.0, mimicking the relatively acidic vesicular pH,and a peak at 370 nm at pH 7.4, the approximate pH of thecytoplasm (Fig. 1C). Although the emission wavelength is in-dependent of pH with a maximum at 453 nm, the intensity ofemission is highly pH dependent (Fig. 1D). Thus, pH can bemeasured ratiometrically via optical means using two differentexcitation wavelengths (15).

Pharmacological Characterization of FFN102. FFN102 was originallyidentified as a vesicular monoamine transporter 2 (VMAT2)substrate using VMAT2-transfected HEK cells (15). The presentimaging studies in mouse brain tissue demonstrate that FFN102 is

Author contributions: P.C.R., D. Sames, and D. Sulzer designed research; P.C.R., D.B.P.,A.B., M.Y.W., C.B., M.S.S., and H.Z. performed research; D. Sames contributed new re-agents/analytic tools; P.C.R., D.B.P., A.B., M.Y.W., M.S.S., H.Z., and D. Sulzer analyzed data;and P.C.R., D.B.P., A.B., D. Sames, and D. Sulzer wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: [email protected] [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1213569110/-/DCSupplemental.

870–875 | PNAS | January 15, 2013 | vol. 110 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1213569110

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also a mouse DAT substrate (see below). FFN102 was purposefullydesigned as a highly polar compound (logD at pH 7.4 is −1.45) todiminish nonselective labeling of tissue and passive membranediffusion. FFN102 showed no appreciable binding to a broad panelof CNS receptors [38 receptors were screened at 10 μM con-centration by the Psychoactive Drug Screening Program (PDSP);Bryan Roth, University of North Carolina, Chapel Hill, NC], in-cluding dopamine (D1–5) and serotonin receptors (5HT1–7) (seeSI Text for a complete list). FFN102 is therefore unlikely to in-terfere with synaptic signaling.

FFN102 Labeling of Dopaminergic Axonal Profiles and PresynapticTerminals in Dorsal Striatum. We examined the uptake and dis-tribution of the probe in mouse brain striatal slices using two-photon microscopy (Fig. 2). We compared the pattern ofFFN102 label with that of green fluorescent protein (GFP)expressed under the control of the tyrosine hydroxylase (TH)promoter (17). A high level of anatomical coincidence betweenFFN102 and TH-GFP staining would indicate selectivity fordopaminergic axonal profiles.Striatal slices from TH-GFP transgenic mice were incubated

with FFN102 (10 μM, 30 min) and imaged sequentially at exci-tation and emission wavelengths corresponding to FFN102 andGFP (FFN102: λexc = 760 nm, λem = 430–470 nm; GFP: λexc = 910nm, λem = 510–580 nm; the two-photon excitation wavelengthused for FFN102 corresponds approximately to the absorptionmaximum at higher pH values, which is 371 nm for one-photonexcitation). The images revealed an extensive overlap (yellow)between GFP (green) (Fig. 2A) and FFN102 labeling (red) (Fig. 2B and C). We found that 91.1 ± 1.9% (mean ± SEM; n = 3) of thepuncta labeled with FFN102 were also labeled with GFP. Toaccount for possible shifts of the field of view in the z plane, thecolocalization of two images of FFN102, acquired before andafter the GFP channel image, was calculated and determined tobe 94.8 ± 0.9% (mean ± SEM; n = 3) (Fig. 2D).We next investigated FFN102 uptake in slice preparations of

mice unilaterally lesioned with the neurotoxin 6-hydroxydop-amine (6-OHDA), which selectively destroys catecholaminergicneurons (18) due to the affinity of the neurotoxin for DAT and

the norepinephrine plasma membrane transporter (NET) (19).Four weeks after the lesion, striatal slices from the lesioned andnonlesioned hemispheres were incubated with FFN102 as de-scribed above. Although 221 ± 10 (mean ± SEM; n = 3)FFN102-positive terminals were present in the nonlesionedhemisphere (right), only 3 ± 2 terminals were identified in thelesioned hemisphere (left) (Fig. 2E). DAT immunolabel con-firmed a loss of dopaminergic terminals in the injected hemi-sphere (Fig. S1). The results thus indicate that FFN102 isselectively accumulated in striatal dopaminergic terminals.

FFN102 Uptake Is Dependent on DAT. We then examined whetheruptake of FFN102 into dopaminergic axonal extensions andterminals was dependent on DAT. Preincubation with the DATinhibitors (20, 21) nomifensine (1 μM, 10 min) or cocaine (5 μM,10 min), followed by a 30-min coincubation of FFN102 (10 μM)with the respective uptake blocker resulted in a 15-fold decreasein the number of labeled terminals (Fig. 3 A and B). Slices notexposed to nomifensine displayed 202 ± 9 terminals (mean ±SEM; n = 3) compared with 12 ± 3 terminals observed in slicespreincubated with the inhibitor. Similarly, slices not exposed tococaine displayed 222 ± 15 terminals (mean ± SEM; n = 3),whereas slices pretreated with cocaine showed only 14 ± 2 ter-minals. Mice with a genetic deletion (“knockout”) of the DATgene (DAT-KO; kindly provided by Marc Caron, Duke Uni-versity, Durham, NC) (22) exhibited a striking decrease in up-take of FFN102 compared with wild-type animals (Fig. 3C).Although 217 ± 7 terminals (mean ± SEM; n = 3) were identi-fied in wild-type slices, only 9 ± 3 terminals were present inDAT-KO slices.As an independent means to test whether FFN102 is a DAT

substrate, we examined the effect of FFN102 on dopaminereuptake during activity-dependent dopamine release using cy-clic voltammetry. As also seen with established DAT blockers(23–25), FFN102 prolonged the decay of the evoked dopaminesignal at concentrations of 4 μM and above (Fig. 3D) (P < 0.05,two-tailed Student t test). The affinity of FFN102 for DAT canbe estimated by making an assumption that it interacts com-petitively with endogenous DA. We previously determined, usinga random walk simulation of amperometric and cyclic voltam-metry signals, that the DA Km-apparent in the striatal slice is ∼0.8 μMand is shifted to larger values by the competitive inhibitors AMPHand nomifensine (25). Similarly, we found that increasing con-centrations of FFN102 progressively shifted the Km-apparent for DAclearance. Ten micromolar FFN102 yielded a shift of Km-apparentto ∼3 μM, consistent with an FFN102 affinity toward DAT of∼4.2 μM: FFN102 thus is a weaker DAT blocker than AMPHor nomifensine (25). In uptake assays using cloned human DATexpressed in HEK293 cells (PDSP), FFN102 inhibited uptake,although its affinity was lower than 10 μM (n = 4) (SI Text).Differences in the affinity estimates may be due to differencesbetween human and mouse DAT or between the assay systems.The ability of FFN102 to function as a substrate for NET was notinvestigated in the slice preparation (hNET binding assay wasnegative) (SI Text).In conclusion, FFN102 is a mouse DAT substrate, as shown by

its DAT-dependent uptake and its ability to inhibit DA uptake.

FFN102 Labeling of Dopaminergic Cell Bodies and Dendrites. Weanalyzed FFN102 uptake in acute midbrain slices comprising thesubstantia nigra (SN) pars compacta and reticulata (Fig. 2 F, H,and I) and the ventral tegmental area (VTA) (Fig. 2 G–I).FFN102 (10 μM; 30–45 min) was accumulated into SN parscompacta and VTA dopamine cell bodies as well as both prox-imal and distal dendrites (Fig. 2 F and G). We occasionally ob-served FFN102 labeling of structures that are likely to be bloodvessels, based on their anatomical characteristics (Fig. 2F). Thevast majority of the cells in the SN pars compacta and VTA la-beled with FFN102 were positive for GFP in the TH-GFP mouseline (Fig. 2 G and H), confirming that FFN102 was selectivelyaccumulated into dopaminergic neurons in these brain regions.

Fig. 1. Structure and photophysical properties of FFN102. (A) Structure ofFFN102. (B) Absorption spectra of FFN102 acquired at a range of pH values(2–10). (C) The excitation spectra of FFN102, measured at an emissionwavelength of 453 nm, displayed a maximum at 340 nm at pH 5.0 (red),whereas a maximum at 370 nm was observed at pH 7.4 (blue). (D) Theemission spectra of FFN102 at both pH 5.0 (red, λex = 340 nm) and pH 7.4 (blue,λex = 370 nm) displayed a maximum at 453 nm.

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Somatodendritic FFN102 accumulation in the midbrain was dueto uptake via DAT, as DAT-deficient mice had no FFN102-la-beled cells (Fig. 3E).Thus, FFN102 shows high selectivity for nigrostriatal and

mesocorticolimbic dopamine neurons, which is primarily as-cribed to its ability to function as a DAT substrate. Moreover,the mostly homogeneous distribution of FFN102 throughout the

cell bodies, dendrites, and projection field terminals of midbraindopamine neurons suggests that the localization of this probe isnot restricted to synaptic vesicles and is also present in thecell cytoplasm.

Release of FFN102 by AMPH. AMPH releases dopamine from bothcytoplasmic and synaptic vesicle pools (23, 26, 27) due to its role

Fig. 2. FFN102 labels dopaminergic terminals in thedorsal striatum and midbrain neurons of acutemouse brain slices. (A) GFP signal (λex = 910 nm)expressed under the control of the TH promoter. (B)Labeling of FFN102 (λex = 760 nm). (C) Overlap ofTH-GFP and FFN102 suggests a high level of coloc-alization (yellow); 91.1 ± 1.9% (mean ± SEM; n = 3)of puncta labeled with FFN102 were also labeledwith TH-GFP. (D) Overlap of two images of FFN102acquired before and after the TH-GFP imageshowed 94.8 ± 0.9% (mean ± SEM; n = 3) colocali-zation. (E) Effect of 6-OHDA lesion on the uptake ofFFN102 in the dorsal striatum of acute mouse brainslices. A statistical difference (P < 0.05, t test; n = 3)in the number of labeled terminals was observed inthe control hemisphere (Right, 221 ± 10; mean ±SEM) compared with the lesioned hemisphere (Left,3 ± 2; mean ± SEM). (Scale bar, 10 μm.) (F) FFN102fluorescent labeling of dopamine neurons in thesubstantia nigra compacta (SNc) and their dendrites(arrows) projecting into the substantia nigra retic-ulata (SNr). The asterisk indicates an apparent bloodvessel. (Scale bar, 50 μm.) (G) FFN102 accumulationin slices from TH-GFP (λex = 930 nm) mice at the levelof the ventral tegmental area (VTA) reveals highdegree of FFN102 and GFP colocalization. (Scale bar,20 μm.) (H) Quantification of FFN102 and GFPcolocalization in SNc and VTA illustrates the pre-dominant labeling of dopaminergic neurons. (I)Schematic illustration modified from Franklin andPaxinos (16) showing the approximate regions atwhich the images were acquired.

Fig. 3. DAT dependency of FFN102 uptake in thedorsal striatum of acute mouse brain slices. (A)Treatment with 1 μM nomifensine decreasedFFN102 uptake (Right, 12 ± 3 puncta; mean ± SEM)compared with untreated slices (Left, 202 ± 9puncta; mean ± SEM). (B) Treatment with 5 μM co-caine resulted in a similar decreased uptake ofFFN102 (Right, 14 ± 2 puncta; mean ± SEM) com-pared with control slices (Left, 222 ± 15 puncta;mean ± SEM). (C) FFN102 uptake was significantlyreduced in the absence of DAT, as seen by thehigher number of fluorescent puncta observed inwild-type slices (WT) (Left, 217 ± 7; mean ± SEM),compared with slices from DAT-deficient mice (DAT-KO; Right, 9 ± 3; mean ± SEM). Values obtained fortreated and untreated slices (A and B), as well asthose obtained for WT and DAT-KO (C), were sta-tistically different (P < 0.05, t test, n = 3). (Scale bar,10 μm.) (D) FFN102 concentrations of 4–40 μMinhibited the reuptake of dopamine released byelectrical stimulation, as measured by cyclic vol-tammetry in the dorsal striatum (P < 0.05, two-tailed Student t test). The height of the signalmostly represents dopamine release, whereas thedecay is mainly dependent on DAT-mediated reup-take. (E) FFN102-positive cells were found in themidbrain (SNc shown) of WT (Left) but not DAT-deficient (Right) mice (n = 3). (Scale bar, 20 μm.)

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as a substrate for both DAT and VMAT2, as well as a weak basecollapsing the interior acidic pH gradients across synaptic vesiclemembranes. We observed a substantial loss of striatal axonalFFN102 fluorescence after perfusion with 1 μM AMPH, whereasin the absence of AMPH, fluorescence of FFN102 decreased ata much slower rate over the same time period (Fig. 4 A–C). Wesimilarly observed relatively slow but appreciable destaining ofsomatodendritic label in control slices (Fig. 4 D–G), with a time-dependent decrease in the rate of fluorescence loss, yieldinga positive value for the subtraction between ΔF1 and ΔF2, thefirst and second 20-min imaging periods (see detailed definitionin legend of Fig. 4 D–F). In AMPH-treated slices, the rate offluorescence loss was increased upon AMPH application duringΔF2, yielding a negative value for ΔF1 − ΔF2 (Fig. 4F) thatdiffered significantly from control slices (P < 0.05, two-tailedt test; Fig. 4 F and G). These data suggest that FFN102 is spon-taneously released from dopaminergic neurons and that AMPHincreases the rate of release. Thus, FFN102 could provide a meansto optically examine the mechanisms involved in AMPH-induceddopamine release.

Activity-Dependent Release of FFN102. Exocytotic release of do-pamine from synaptic vesicles can be evoked by neuronal de-polarization with a high concentration of potassium or by electricalstimulation. Perfusion of FFN102-labeled slices with artificialcerebrospinal fluid (ACSF) containing 40 mM KCl for 5 minresulted in the loss of fluorescent signal (760-nm excitation)from presynaptic terminals and an overall increase in back-ground fluorescence (Fig. S2B). The increased background isexpected from the effect of pH on FFN102’s signal, as uponexocytosis, FFN102 would be redistributed from the acidic en-vironment of the synaptic vesicle lumen to the neutral extracel-lular space, resulting in increased fluorescence at 760 nm. Therelease of FFN102 in response to K+ depolarization was blockedby cadmium chloride (CdCl2) (200 μM), a calcium channelblocker (Fig. S2C), whereas the loss of FFN102 in the presenceof KCl and CdCl2 was not different from that observed undercontinuous ACSF perfusion in the same time period (Fig. S2 Aand C). These results demonstrate that FFN102 is released inresponse to depolarization in a Ca2+-dependent manner.

We then investigated the exocytotic release of FFN102 usinglocal electrical stimulation to control the frequency and numberof pulses applied. Slices were imaged for 140 s without stimu-lation, and for an additional 300 s period during which a 10-Hzstimulus train was locally applied with a bipolar electrode.Within 10–40 s of the onset of stimulation, an increase in fluo-rescence intensity measured at the puncta was observed (Fig.5A). An even greater relative increase was observed in areas thatexcluded the puncta (“background”), indicating that this fluo-rescence increase was due to diffusion of the probe from pre-synaptic release sites (Fig. 5B). No significant increase in punctaor background fluorescence was observed when voltage-gatedcalcium channels were blocked by 200 μM CdCl2 or in the ab-sence of stimulation. Because the fluorescence emitted by theprobe present in the extracellular space is likely to contribute tothe overall fluorescence intensity measured at the puncta, due toboth light scattering and insufficient z resolution of two-photonmicroscopy, we subtracted average background fluorescencevalues from the total fluorescence signals measured at eachpunctum (Fig. 5C). The background subtraction removed theincrease in fluorescence measured at fluorescent puncta in re-sponse to stimulation (Fig. 5C). These results indicate that theincreased fluorescence upon stimulation is mainly due to an in-crease in background fluorescence values, reflecting the releaseof reporter from acidic synaptic vesicles into the neutral extra-cellular space where the fluorescence intensity of FFN102 at 760-nm excitation is enhanced.Background subtraction also uncovered a difference (P <

0.001, ANOVA) in the rate of puncta fluorescence loss betweenstimulated and nonstimulated slices, indicating that stimulation-induced destaining of fluorescent puncta had occurred (Fig. 5C).Upon examination of background-subtracted curves from in-dividual fluorescent puncta, we found that stimulated slicesexhibited both destaining and nondestaining puncta within thesame field of view. Representative examples of destaining andnondestaining curves are shown in Fig. 5D. This finding impliesthat averaging the fluorescence intensities of all puncta, as shownin Fig. 5C, underestimates the fluorescence loss of destainingpuncta elicited by electrical stimulation. In an effort to betterrepresent the different responses of FFN102 puncta, we grouped

Fig. 4. AMPH induces release of FFN102 from dorsalstriatum and midbrain. (A and B) Images of an acutestriatal slice loaded with FFN102 taken 0 and 10 minafter perfusion with ACSF containing no AMPH (A) or1 μM AMPH (B). (C) Quantification of FFN102 punctafluorescence loss in the absence and presence of1 μM AMPH, normalized to the intensity at time0 (expressed as mean ± SEM; n = 3). (D) Schematic il-lustration of midbrain AMPH-induced FFN102 releaseexperiments. FFN102-loaded midbrain slices wereperfused with ACSF for 15 min in the imaging cham-ber and images were acquired 0, 20, and 40 minthereafter. One-half of the slices were perfused withACSF containing 10 μM AMPH during the 20- to 40-min period, whereas the other half was perfused withregular ACSF throughout the entire experiment. (E)Representative images of FFN102-filled SNc DA neu-rons acquired after 0, 20, and 40 min of ACSF (Upper)or ACSF plus 10 μM AMPH (Lower) perfusion. (F)Quantification of fluorescence intensity shows a de-creased rate of fluorescence loss between the 20- and40-min time points (ΔF2) compared with the 0- to 20-min period (ΔF1) in control slices, but increased rate inAMPH-treated slices. (G) Fluorescence over time afternormalization. Note the more rapid decrease in fluo-rescence in the presence of AMPH. *P < 0.05 com-pared with control slices (t test; n = 28–29 cells from 6slices per treatment). For F and G, results are displayedas mean ± SEM. (Scale bar: A and B, 10 μm; E, 20 μm.)

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the destaining and nondestaining puncta into separate cohortsand produced average intensity plots depicted in Fig. 5E (seealso Fig. S3). Destaining puncta responded to stimulation withan exponential decay of fluorescence (Fig. 5 D and E) witha mean destaining half-time (t1/2) of 88.0 ± 9.4 s (mean ± SEM).The distribution of t1/2 values obtained for all individualdestaining puncta is shown as a histogram in Fig. 5F. A smallfraction of destaining puncta in stimulated slices appeared toshow either a transient increase in fluorescence intensity fol-lowed by destaining or a lag period between the onset of stim-ulation and destaining. These kinetics, which may reflect forexample incomplete background subtraction (SI Text), did notfollow a clear exponential decay upon stimulation, and, there-fore, these puncta were not included in the analysis or the av-erage data presented in Fig. 5E. All puncta considered asdestaining presented R2 exponential fitting values of 0.6 orhigher (SI Text). Puncta considered as nondestaining displayedthe same slow loss of signal as puncta from nonstimulated con-trols or from slices exposed to 10-Hz stimulation in the presenceof CdCl2. The time course observed for the transient increase inbackground fluorescence mentioned above approximately matchedthe kinetics of fluorescence loss of the destaining puncta, which isconsistent with an activity-dependent exocytotic release of FFN102.Our results demonstrate that FFN102 enables optical detection ofthe overall release of synaptic content into the extracellular spaceas well as examination of release kinetics of individual presynapticterminals in striatal slices.

DiscussionWe recently introduced FFNs as optical probes to visualizeneurotransmission at the level of individual presynaptic termi-nals, as well as pH-dependent FFNs that can be used to de-termine pH of VMAT1-containing vesicles in PC12 cells (14, 15).FFN102 is to date the most selective probe for dopamine pre-synaptic terminals in mouse acute brain tissue and can be used tovisualize DAT activity. FFN102 can in addition be used forfunctional studies in somatodendritic regions of dopamine neu-rons as well as to address synaptic activity of individual dopa-minergic terminals. Furthermore, in contrast to the firstgeneration of FFNs that only permitted measuring the levels ofFFN that remain in presynaptic terminals, the reporter FFN102provides an approach to observe both the FFN that is releasedinto the extracellular milieu and the label remaining withinindividual terminals.The high selectivity of this probe in the brain slice can be at-

tributed to its ability to function as a DAT substrate and to its

high polarity. Moreover, the lack of interaction of FFN102 witha broad panel of CNS receptors bodes well for the use ofFFN102 to study presynaptic activity and plasticity, diminishingpotential concerns in data interpretation due to modulation ofneurotransmitter receptors. Although FFN102 accumulation instriatal slices was clearly DAT dependent, we were unable toobserve FFN102 uptake in dopamine cell culture, possibly due tolower DAT or VMAT2 levels in cultured postnatal neurons.Other fluorescent monoamine transporter substrates have

been developed to visualize multiple aspects of monoamineneuron function. For example, the fluorescent serotonin (5HT)analog 5,7-dihydroxytryptamine (5,7-dHT) can identify 5HT anddopamine neurons in culture due to its uptake by serotonintransporter (SERT) and DAT, respectively. 5,7-dHT has re-cently been applied to study somatic release of 5HT from sero-tonergic neurons in brain slice preparations (28). However, thisprobe is weakly fluorescent, prone to oxidation and toxic. Fur-thermore, 5,7-dHT vesicular uptake requires long incubationtimes (3 h) in the presence of high concentration of monoamineoxidase inhibitors, which renders this probe less suited forphysiological studies of synaptic plasticity in brain tissue. Incontrast, FFN102 exhibits rapid uptake (within 30 min) and ischemically and photochemically stable, highly fluorescent, andproduced no apparent toxicity in in vitro and in situ studies.These properties render this agent suitable for time-lapse two-photon microscopy imaging of DAT function and neurotrans-mitter release. Similarly, NET activity at murine sympatheticterminals has been measured optically using a fluorescent NET/DAT/SERT substrate present in the commercially availableNTUA systems (neurotransmitter transporter uptake assay) (29).However, it is currently unknown whether this system can beused for studies of synaptic activity in brain tissue.The loss of striatal terminal FFN102 elicited by AMPH is

reminiscent of the effect of this psychostimulant in the re-distribution of endogenous dopamine. However, AMPH-inducedrelease of dopamine and FFN102 proceeds with different timecourses. Cyclic voltammetry recordings indicate that AMPH-induced dopamine release reaches a plateau after 30 min (23). Incontrast, we observed an almost complete release of FFN102 inresponse to AMPH within 10 min. Interestingly, AMPH-induceddopamine efflux becomes faster upon VMAT2 inhibition (23),which likely reflects the time course of reverse transport of cy-toplasmic dopamine. The slower release measured in the ab-sence of VMAT2 inhibitors is probably due to the redistributionof vesicular dopamine to the cytosol that precedes efflux viareverse transport through DAT. We thus hypothesize that

Fig. 5. Calcium-dependent release of FFN102 inthe dorsal striatum of acute mouse brain slices inresponse to electrical stimulation. (A–C) Normalizedmean fluorescence intensity of fluorescent puncta(A), regions that do not include the puncta, i.e.,“background” (B), and fluorescent puncta afterbackground subtraction (C). Mean values are shownfor unstimulated slices (red) and slices stimulated at10 Hz in the absence (blue) or presence of 200 μMCdCl2 (green). Fluorescence intensities for eachcurve were normalized to the corresponding in-tensity values at the time point before the onset ofstimulation (t = 0). Each point represents a mean ±SEM (n = 5). (D) Relative fluorescence over time ofrepresentative individual destaining (blue) andnondestaining terminals (black). For calculation ofhalf-time (t1/2) values, each curve was fit with a one-phase exponential decay function. For the expo-nential curves shown, t1/2 values were calculated tobe 59.5, 76.5, 100.4, 146.3, and 310.3, with R2 valuesof 0.89, 0.97, 0.92, 0.93, and 0.95, respectively. (E)Relative signal over time of destaining (blue) andnondestaining puncta (black), after background subtraction, in slices stimulated at 10 Hz. Each curve represents the mean ± SEM (n = 5). For A–E, stimulationwas initiated at time 0 s. (F) Histogram of t1/2 values of individual FFN102-labeled terminals that destain under 10-Hz stimulation. (Bin size, 20 s.)

874 | www.pnas.org/cgi/doi/10.1073/pnas.1213569110 Rodriguez et al.

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a significant fraction of FFN102 is localized in the cytosol. This isfurther supported by the high correlation of FFN102 and TH-GFP labeling (Fig. 2) including both apparent presynaptic termi-nals and axonal regions that do not include clear puncta (Fig. S4),and also by the apparently homogeneous distribution of FFN102within cell bodies and dendrites (Fig. 2).Ca2+-dependent release of FFN102 elicited by high-K+ or

electrical stimulation was detected in striatal terminals as (i) anoverall increase in background signal, reflecting the release ofFFN102 during vesicle fusion from acidic synaptic vesicles to theneutral extracellular space and (ii) a decrease in fluorescence ofindividual puncta. Analysis of the response of individual fluo-rescent puncta to a 10-Hz stimulation train revealed the presenceof destaining and nondestaining puncta. The background-sub-tracted fluorescence intensity of destaining puncta exhibited anexponential decay upon the onset of stimulation with a mean t1/2of 88.0 ± 9.4 s. Nondestaining puncta may either represent axonalareas that do not contain synaptic vesicles but contain FFN102 inthe cytosol or dopaminergic terminals that do not undergo de-tectable synaptic vesicle exocytosis in response to stimulation.Further studies combining the use of FFNs and probes thatmeasure vesicle fusion are underway to address this issue.In conclusion, FFN102 is a bright fluorescent probe compati-

ble with a range of red and green fluorescent markers (e.g., GFP,FM dyes) that exhibits high selectivity toward dopamine neurons.For this reason, FFN102 is a promising tool to visualize neuronaldegeneration in the 6-OHDA and other Parkinson’s diseasemodels and to study important functional parameters such asDAT activity, somatodendritic dopamine dynamics, dopamineexocytosis at individual presynaptic terminals, and mechanismsunderlying presynaptic plasticity and the action of drugs of abuse.

MethodsSlice Preparation. Unless otherwise noted, all animals used for slice prepa-ration were 2- to 4-month-old male C57BL/6 mice obtained from The JacksonLaboratory. All animal protocols were approved by the Institutional AnimalCare and Use Committee of Columbia University.

For striatal slice preparation, micewere decapitated and acute 300-μm-thickcoronal slices were cut on a vibratome and allowed to recover for 1 h beforeuse at room temperature in oxygenated [95% O2, 5% CO2 (vol/vol)] ACSF

containing the following (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 0.3 KH2PO4,2.4 CaCl2, 1.3 MgSO4, 0.8 NaH2PO4, 10 glucose (pH 7.2–7.4, 292–296 mOsm/L).

For coronal midbrain slice preparation, brains were dissected and im-mersed in 4 °C cutting solution containing the following (in mM): 180 su-crose, 10 NaCl, 2.5 KCl, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 10glucose (pH 7.3, 300–305 mOsm/L). Coronal sections (250 μm) were cutthrough the midbrain and equilibrated for 30 min in 32 °C ACSF. Slices werethen transferred to room temperature (22–25 °C) for 30 min before use.

Loading and Imaging of FFN102. FFN102 (10 μM) (available from AbcamBiochemical) was loaded into presynaptic terminals by a 30- to 45-min in-cubation at room temperature in oxygenated ACSF. Slices were then trans-ferred to an imaging chamber (QE-1; Warner Instruments), held in placewith a platinum wire and nylon custom-made holder (30), and superfused(1–3 mL/min) with oxygenated ACSF. Slices were allowed to wash in theperfusing chamber for 5–10 min before imaging.

Unless otherwise noted, dopaminergic terminals and dopamine cell bodieswere visualized at >30-μm depth in the slice using either a Prairie UltimaMultiphoton Microscopy System (Prairie Technologies) or a Leica DM6000with titanium–sapphire MaiTai lasers (Spectra-Physics) equipped with either60×, 0.9 N.A., or 40×, 0.8 N.A. water-immersion objectives. FFN102 was ex-cited at 760 nm and visualized using an emission range of either 440–500 nm(Prairie) or 430–470 nm (Leica).

Please refer to SI Text for methodology regarding FFN102 photophysicalcharacterization, 6-OHDA injections, cyclic voltammetry recordings, FFN102-GFP colocalization experiments, effects of drugs, KCl, and electrical stimulationon FFN102 loading and/or destaining, and image processing and analysis.

ACKNOWLEDGMENTS. We thank Paolomi Merchant and Matthew Dunn forexperimental support, the National Institute of Mental Health (NIMH)Psychoactive Drug Screening Program (University of North Carolina, ChapelHill) for pharmacological characterization, Marc Caron (Duke University) forproviding the DAT knockout mice, and Eugene Mosharov (ColumbiaUniversity) for ImageJ macros. This work was supported by The G. Haroldand Leila Y. Mathers Charitable Foundation, NIMH Grants R01MH086545and R21 MH090356, the JPB Foundation, the McKnight Foundation, theParkinson’s Disease Foundation, National Institute on Drug Abuse GrantR01DA07418, and National Institute of Neurological Disorders and StrokeUdall Center of Excellence for Parkinson’s Disease Research. P.C.R. was sup-ported by the National Science Foundation predoctoral fellowship. A.B. wassupported by fellowships from the Swedish Research Council and theSweden–America Foundation.

1. Wise RA (2004) Dopamine, learning and motivation. Nat Rev Neurosci 5(6):483–494.2. Verhoeff NP (1999) Radiotracer imaging of dopaminergic transmission in neuropsy-

chiatric disorders. Psychopharmacology (Berl) 147(3):217–249.3. Nikolaus S, Antke C,Müller H-W (2009) In vivo imaging of synaptic function in the central

nervous system: I. Movement disorders and dementia. Behav Brain Res 204(1):1–31.4. Nikolaus S, Antke C, Müller H-W (2009) In vivo imaging of synaptic function in the

central nervous system: II. Mental and affective disorders. Behav Brain Res 204(1):32–66.

5. Sotnikova TD, Beaulieu J-M, Gainetdinov RR, Caron MG (2006) Molecular biology,pharmacology and functional role of the plasma membrane dopamine transporter.CNS Neurol Disord Drug Targets 5(1):45–56.

6. Khan SH, Shuaib A (2001) The technique of intracerebral microdialysis.Methods 23(1):3–9.

7. Zhang M-Y, Beyer CE (2006) Measurement of neurotransmitters from extracellularfluid in brain by in vivo microdialysis and chromatography-mass spectrometry. JPharm Biomed Anal 40(3):492–499.

8. Stamford JA (1989) In vivo voltammetry—prospects for the next decade. TrendsNeurosci 12(10):407–412.

9. Robinson DL, Venton BJ, Heien MLAV, Wightman RM (2003) Detecting subseconddopamine release with fast-scan cyclic voltammetry in vivo. Clin Chem 49(10):1763–1773.

10. Mosharov EV, Sulzer D (2005) Analysis of exocytotic events recorded by amperometry.Nat Methods 2(9):651–658.

11. Gaffield MA, Betz WJ (2006) Imaging synaptic vesicle exocytosis and endocytosis withFM dyes. Nat Protoc 1(6):2916–2921.

12. Miesenböck G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptictransmission with pH-sensitive green fluorescent proteins. Nature 394(6689):192–195.

13. Li Z, et al. (2005) Synaptic vesicle recycling studied in transgenic mice expressingsynaptopHluorin. Proc Natl Acad Sci USA 102(17):6131–6136.

14. Gubernator NG, et al. (2009) Fluorescent false neurotransmitters visualize dopaminerelease from individual presynaptic terminals. Science 324(5933):1441–1444.

15. Lee M, Gubernator NG, Sulzer D, Sames D (2010) Development of pH-responsivefluorescent false neurotransmitters. J Am Chem Soc 132(26):8828–8830.

16. Franklin KBJ, Paxinos G (1997) The Mouse Brain in Stereotaxic Coordinates (AcademicPress, San Diego).

17. Sawamoto K, et al. (2001) Visualization, direct isolation, and transplantation ofmidbrain dopaminergic neurons. Proc Natl Acad Sci USA 98(11):6423–6428.

18. Ungerstedt U (1968) 6-Hydroxy-dopamine induced degeneration of central mono-amine neurons. Eur J Pharmacol 5(1):107–110.

19. Jonsson G, Sachs C (1971) Uptake and accumulation of 3H-6-hydroxydopamine inadrenergic nerves. Eur J Pharmacol 16(1):55–62.

20. Torres GE, Gainetdinov RR, Caron MG (2003) Plasma membrane monoamine trans-porters: Structure, regulation and function. Nat Rev Neurosci 4(1):13–25.

21. Brogden RN, Heel RC, Speight TM, Avery GS (1979) Nomifensine: A review of itspharmacological properties and therapeutic efficacy in depressive illness. Drugs 18(1):1–24.

22. Giros B, Jaber M, Jones SR, Wightman RM, Caron MG (1996) Hyperlocomotion andindifference to cocaine and amphetamine in mice lacking the dopamine transporter.Nature 379(6566):606–612.

23. Jones SR, Gainetdinov RR, Wightman RM, Caron MG (1998) Mechanisms of amphet-amine action revealed in mice lacking the dopamine transporter. J Neurosci 18(6):1979–1986.

24. Schmitz Y, Benoit-Marand M, Gonon F, Sulzer D (2003) Presynaptic regulation ofdopaminergic neurotransmission. J Neurochem 87(2):273–289.

25. Schmitz Y, Lee CJ, Schmauss C, Gonon F, Sulzer D (2001) Amphetamine distortsstimulation-dependent dopamine overflow: Effects on D2 autoreceptors, trans-porters, and synaptic vesicle stores. J Neurosci 21(16):5916–5924.

26. Sulzer D, Sonders MS, Poulsen NW, Galli A (2005) Mechanisms of neurotransmitterrelease by amphetamines: A review. Prog Neurobiol 75(6):406–433.

27. Sulzer D, et al. (1995) Amphetamine redistributes dopamine from synaptic vesicles tothe cytosol and promotes reverse transport. J Neurosci 15(5 Pt 2):4102–4108.

28. Colgan LA, Putzier I, Levitan ES (2009) Activity-dependent vesicular monoaminetransporter-mediated depletion of the nucleus supports somatic release by serotoninneurons. J Neurosci 29(50):15878–15887.

29. Parker LK, Shanks JA, Kennard JA, Brain KL (2010) Dynamic monitoring of NET activityin mature murine sympathetic terminals using a fluorescent substrate. Br J Pharmacol159(4):797–807.

30. Wong MY, Sulzer D, Bamford NS (2011) Imaging presynaptic exocytosis in cortico-striatal slices. Methods Mol Biol 793:363–376.

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