Single-vesicle imaging reveals that synapticvesicle exocytosis and endocytosis arecoupled by a single stochastic modeJ. Balaji and T. A. Ryan*

Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065

Edited by Francisco Bezanilla, University of Chicago, Chicago, IL, and approved November 6, 2007 (received for review August 10, 2007)

The nature of synaptic vesicle recycling at nerve terminals has beena subject of considerable debate for >35 years. Here, we report theuse of an optical strategy that allows the exocytosis and retrievalof synaptic components to be tracked in real time at single-molecule sensitivity in living nerve terminals. This approach hasallowed us to examine the recycling of synaptic vesicles in responseto single action potentials. Our results show that, after exocytosis,individual synaptic vesicles are retrieved by a stochastic processwith an exponential distribution of delay times, with a mean timeof �14 s. We propose that evidence for fast endocytosis, such asthat proposed to support the presence of kiss-and-run, is likelyexplained by the stochastic nature of a slower process.

kiss-and-run � endocytic dwell time � pHluorin

Once synaptic vesicles undergo exocytosis and release neuro-transmitter, they are recycled for further rounds of reuse.

Although significant evidence indicates synaptic vesicle endocytosisis dynamin- (1–3) and clathrin- (4) mediated, a number of studieshave also implicated a mechanism of direct vesicle retrieval thatwould bypass the more classical clathrin-mediated route. In general,evidence for a kiss-and-run pathway is based on experiments thatpostulate a faster mechanism of retrieval than is usually reported forclathrin-mediated endocytosis. The first such evidence arose in theoriginal experiments in bipolar nerve terminals, where electricalcapacitance recordings revealed an endocytic time constant of �2s (5). Experiments in small CNS nerve terminals include use of FMdyes reporting that, under certain conditions, dye does not com-pletely destain during an exocytosis and retrieval event (6–9). Thesedata have been interpreted as evidence for retrieval events hap-pening in �1 s, much faster than has been reported for theclathrin-mediated route. More recent data have suggested that suchevents predominate in hippocampal neurons for a stimulus fre-quency at 0.1 Hz (10). In addition, electrical capacitance measure-ments at a giant calyceal synapse have provided evidence thatsynaptic vesicle retrieval can happen on a second or even subsecondtimescale (11), however only for a relatively small fraction of events.Although these studies point to the possibility that some retrievalevents might occur on a �1 sec timescale, it is unclear whether suchevents represent a separate pathway or simply one end of adistribution of times of a common process. We propose that themere presence of faster events does not necessarily imply that aseparate mode of retrieval is operational.

To determine whether multiple modes of retrieval are present,we sought to examine the distribution of retrieval times for all eventsafter exocytosis. Here, we report an improved approach thatcombines specific targeting of pHluorin, a GFP with modified pHsensitivity (12, 13), with high collection-efficiency optical detectionthat allows one to follow exocytosis and endocytosis of individualsynaptic vesicles. Targeting of pHluorin to synaptic vesicles byfusion to an intraluminal loop of the vesicular glutamate transportervGlut1 (14) resulted in much lower surface expression. The result-ing decrease in background fluctuations provided a much highersignal-to-noise measurement than has previously been possible.This approach allows for the direct detection of fusion and retrieval

of single synaptic vesicles at individual nerve terminals and indicatesthat a stochastic mode couples exocytosis with synaptic vesicleretrieval.

ResultsImprovements in Optical Recordings. Detection of single-vesicleexocytosis using pHluorins has been limited by a combination offluctuations in baseline fluorescence arising in part from a fractionof synaptic vesicle proteins residing on the synaptic surface (13, 15),photobleaching, as well as the Johnson noise and quantum effi-ciency of the photodetector. Of the approximately eight possiblechoices of synaptic vesicle proteins (16, 17) for targeting pHluorinto the synaptic-vesicle lumen, those tested to date have significantsurface expression of the pHluorin-tagged molecule at the synapse[vesicle-associated membrane protein (VAMP) �15% (13), syn-aptotagmin I �22% (15), synaptophysin �9% (4)]. The vesicularglutamate transporter vGlut1 was recently tagged successfully withpHluorin, and expression of this construct does not appear to alterpresynaptic function (14). To estimate the surface fraction ofvGlut1-pHluorin (vGpH) after transfection into neurons, we usedacute applications of NH4Cl to rapidly alkalinize the acidic internalpools of the molecule without altering surface fluorescence (13).These experiments indicate that transfection of this constructresults in only �2% surface expression at synapses (Fig. 1 A–C),indicating that vGlut1 resides largely in synaptic vesicles underresting conditions, and it is a good candidate for optimizing thedetection of small signals using pHluorins. To optimize vGpHfluorescence detection, we used detectors suited for single-molecule imaging, with �95% quantum efficiencies in the visiblespectrum and Johnson noise suppressed to well below one electronper readout as the result of on-chip electron multiplication (18).Our optical scheme consisted of coupling laser-excited fluorescenceto this detector with minimal optical elements in the path. Wedetermined the sensitivity of our optical system using a dilutesolution of immobilized EGFP molecules on a coverslip where theyappear as diffraction limited spots (Fig. 1 D and F). Analysis of theintensity distribution of these spots revealed a quantized distribu-tion with a unitary size of �58 � 3.3 units (Fig. 1G), which weattribute to the fluorescence of a single EGFP molecule. Weexamined the intensity changes after photobleaching by intenseillumination (Fig. 1E) to verify that these spots corresponded tosingle molecules. As expected for single fluorophores, photobleach-ing occurred in an all-or-none fashion, where the histogram offluorescence changes (Fig. 1H) was well described by the sum ofdistributions with peaks at zero intensity (no bleaching), �64(single-molecule bleaching), and at �134 (two-molecule bleaching)fluorescence units. We attribute the center of the second peak as

Author contributions: J.B. and T.A.R. designed research; J.B. performed research; J.B. andT.A.R. analyzed data; and J.B. and T.A.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

*To whom correspondence should be addressed. E-mail: taryan@med.cornell.edu.

© 2007 by The National Academy of Sciences of the USA

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corresponding to the mean intensity of single EGFP molecules,which is in good agreement with the quantal size determined beforebleaching (Fig. 1G Inset). In addition to demonstrating the overallsensitivity of the optical system, these measurements also providea means of calibrating the experiments with appropriate gain andillumination corrections in terms of number of EGFP molecules.

Detection of Exocytosis and Endocytosis in Response to Single ActionPotential (AP) Stimuli. Under these experimental conditions, syn-apses expressing vGpH showed robust fluorescence responses tosingle AP stimulation (Fig. 2 A and B) with photobleaching rates ofonly �0.01% per frame (see Materials and Methods). The ensembleaverage of the fluorescence change driven by single stimuli (Fig. 2C)shows a sharp rise followed by a much slower decay. Signals derivedfrom pHluorins are based on deprotonation of GFP during thefusion event and a pH transition from �5.5 to 7.4. After endocy-

tosis, reacidification of the vesicle lumen quenches the fluorescence.The ensemble average shown here is well described by a simplekinetic model of two sequential first-order processes correspondingto endocytosis and reacidification (ref. 4; see Materials and Meth-ods). Using rapid quenching approaches, vesicle reacidification hasrecently been estimated to occur with a time constant of �reacid �4 s (4, 16). The time constant for endocytosis derived from fittingthe data in Fig. 2C indicates that �endo � 15 � 1 s.

During low-frequency repetitive stimulation, on-going endo-cytosis and reacidification prevent continuous increase in fluo-rescence (Fig. 2D, black trace). We verified that the fluorescencedecay after each stimulus corresponds to endocytosis and re-acidification, because these decays were completely blocked bythe V-ATPase inhibitor bafilomycin (19) and instead showedstaircase-like fluorescence increases in response to continuousstimulation (Fig. 2D, red trace). In addition, these observations

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Fig. 1. Improvements in pHluorin detection. (A and B) Images of the vGlut1-pHluorin-transfected hippocampal boutons before and after addition of NH4Cl.(C) The ensemble average of the fluorescence intensity at boutons indicates a �17-fold increase in fluorescence corresponding to a surface fraction of 2 � 0.8%assuming an initial vesicular pH � 5.5 (nine neurons, �50 boutons each). (D) Fluorescence intensity of single EGFP molecules. Smoothened surface plot (3 � 3)of GFP in polyacrylamide gel before and after bleaching is shown in D and E, respectively. (F) Intensity profile of a fluorescence spot (circles). A Gaussian fit (line)yields a FWHM of 2.8, in excellent agreement with point spread function measured from 100-nm beads in our setup. (G) Intensity distribution of fluorescentpuncta before bleaching is well fit (reduced �2 � 0.98) by a set of Gaussians whose spacings are exact integer multiples (Inset, linear fit, slope � 58 � 3 fluorescenceunits per peak, R � 0.998). (H) Distribution of the change in fluorescence of the puncta after bleaching �F (Fprebleach � Fpostbleach) (bars) shows peaks at 0, 64, and134. Solid line is the overall fit, and dotted lines are the individual Gaussian fits.

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allow us to determine whether any exocytosis events might befollowed by endocytosis and reacidification that is faster than ourtime resolution. We reasoned that any vesicle that fuses andreleases its protons would still require the V-type ATPase toreacidify the lumen, regardless of the speed of endocytosis. Thus,if such events occur, they would be trapped in the fluorescentstate in the presence of bafilomycin. The amplitude of the

average fluorescence response to a single action potential(�F1AP) in the presence of bafilomycin should, in turn, be largerthan that in the absence of bafilomycin. Comparison of thecumulative intensity histograms of �F1AP values obtained withand without bafilomycin across many boutons showed that thedistributions were indistinguishable (Fig. 2E), indicating thoseevents must happen only very rarely. Thus, these measurementsare not biased by missing exocytosis events that subsequentlyendocytose and reacidify too rapidly to be detected, and weconclude that all fusion events that lead to the release of protonscontribute to the signal.

Analysis of AP-Driven Fluorescent Transients from Single Boutons. Wesought to characterize the distribution of signals associated withvGpH responses at individual boutons after single APs. Examplesof fluorescence traces from individual boutons stimulated withsingle APs at 20-s intervals are shown in Fig. 3A. These data indicatethat fluorescence increases at single boutons occur as suddenstep-like changes, as would be expected for exocytosis (Fig. 3A).The distribution of fluorescence changes resulting from single APs(�F1AP) derived from repeated trials (spaced �2 min apart) atmany boutons (Fig. 3B) is well fit (reduced �2 � 0.97) by a set ofGaussians with different means and areas. The best fit, however,yielded a set of Gaussians with exact-integer multiple spacing (Fig.3 B and C) that is insensitive to bin width (see Materials andMethods). The first peak of the distribution, centered about zerointensity, reflecting events that failed to show any exocytosis, issimilar to the noise peak measured in the absence of stimulation(Fig. 4A). All other peaks represent multiples of a discrete quantalsize. If this distribution truly reflects the variation from trial to trialof quantal responses, we presume that the area under each peak,and not the spacing, should vary as release probability is changed.We tested this hypothesis by comparing the event size distributionfor trials performed in 1.5 mM external CaCl2 ([Ca]e) with thatperformed in 4 mM [Ca]e. At lower [Ca]e, the distribution shows amuch larger peak around zero amplitude, whereas independent fitsto the two data sets revealed identical quantal size (Fig. 4 A and B).Our data thus show that single AP responses at single boutons arisein discrete steps that are multiples of a basic size. It is possible thathigher-order peaks reflect fusion events of vesicles with a greaternumber of vGpH; however, that these peaks appear only underconditions of elevated release probability argues against this pos-sibility. The data obtained at lower release probability ([Ca]e � 1.5

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Fig. 2. Optical detection of single-vesicle responses. Image of vGlut1-pHluorin-transfected hippocampal boutons before (A) and after (B) applyinga one-AP stimulus. Arrowheads point to some of the responding boutons.(Scale bar: 10 �m.) (C) The gray line is the average of single action potentialresponse over 120 events. The black line is the fit to a double exponentialdescribing endocytosis followed by reacidification (see Materials and Meth-ods). The fit yields a time constant for endocytosis of 15 � 1 s. (D) In thepresence of bafilomycin (red trace), repeated stimulation at 0.2 Hz results ina continuous staircase increase in fluorescence, whereas in the control run(black trace), endocytosis and reacidification balance the exocytic-driven in-crease. (E) The cumulative histogram of the response amplitudes to single APin the absence (squares) and presence (circles) of bafilomycin (three neurons,165 boutons) are identical (Kolmogorov–Smirnov test maximal D value �0.1,which corresponds to P � 1.000 that the distributions are the same), indicatingall fusion events that release protons contribute to the signal.

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Fig. 3. Distribution of instantaneous fluorescence changes. (A) Representative fluorescence time traces of individual boutons during repeated stimulation(indicated by arrows). An example of the amplitude of the first event (�F1AP) is shown in the top upper left trace. (B) The distribution of instantaneous fluorescencechange amplitudes from individual boutons associated with single-AP stimuli (�F1AP) shows integer multiples of quantized fluorescence intensities. The data (540events) were obtained from 30 boutons in 1.5 (four trials), 2 (four trials), 2.5 (three trials), 3.5 (three trials), and 4 mM (four trials) CaCl2. The solid and dottedblack lines are the overall and individual fits to multiple Gaussians, respectively (reduced �red

2 � 0.97). The coefficient of variation for the first nonzero peak is0.37. (C) The peak positions obtained as a fit parameter in B are integer multiples indicating that �F1AP are distributed as multiples of a fundamental quantalunit (r � 0.998).

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mM) allows one to set an upper bound on the number of eventsattributable to brighter vesicles by comparing the area under thefirst and second nonzero peaks. These data indicate that at least80% of individual vesicles undergoing fusion have a fluorescenceintensity that corresponds to the first nonzero peak in the distri-bution, and thus higher-order peaks largely correspond to the fusionof multiple vesicles at the same bouton. Examples of fusion eventscorresponding to the median of the first (one vesicle) and sec-ond peaks (two vesicles) compared with a noise trace are shown inFig. 4C.

The quantal size expressed in fluorescence units can be directlycompared with the intensity of single EGFP molecules (Fig. 1 G andH). Comparison of the fluorescence intensity of the quantal sizeusing vGpH (Fig. 3C) with that of single immobilized EGFPmolecules indicates that a single EGFP under these conditionscorresponds to 1.23 � 0.1 vGpH quanta. Thus, single vGpH quantacorrespond to near single-molecule events. That a single vGpHquantum did not correspond to an exact integer number of EGFPfluorescence equivalents is possibly due to the difference in envi-ronment of immobilized EGFP compared with pHluorin splicedinto vGlut1 and expressed on the cell surface. Thus, although theabsolute calibration does not provide a perfect one-to-one corre-spondence between single EGFP signal amplitudes and vGpHquanta, the comparison indicates that the quantal size likely cor-responds to very few, if not single, vGpH molecules. We have alsoobtained estimates of the total number of vesicles labeled bycomparing the intensity of the maximal fluorescence obtainedduring sustained stimulation at high frequency in the presence of

bafilomycin with that of our first nonzero peak in the quantaldistribution. This type of stimulation results in the entire recyclingpool becoming alkaline (19) and indicates that, on average, thelabeled pool size was �64 � 14 vesicles (n � 4 experiments, �300total boutons). This is very similar to estimates obtained based onFM labeling (not shown), indicating that most recycling vesicleslikely contain a single vGlut-pHluorin, implying the sorting processfor insertion of the reporter into the vesicle is not random.

Endocytosis Is Well Described by a Single Poisson Process. Thefluorescence decay of individual endocytic events probed withpHluorin should correspond to two sequential steps: a dwell timeon the cell surface (the time for endocytosis) followed by vesiclereacidification. We characterized the timescale of endocytic re-trieval of single vesicles by restricting the analysis to fusion eventswhose intensity amplitude was within 1 SD of the center of the peakin Fig. 3B. This restriction is necessary, because multiple vesicleswould potentially endocytose at different times, resulting in com-plex fluorescence decays. Examples of the time course of single-vesicle events are shown in Fig. 5A and reveal that the decay phaseoccurs asynchronously with respect to the stimulus. pHluorinmolecules report a time-averaged ensemble of pH, because thedissociation time for protons is in the millisecond time range (20);thus, even for individual events, the time course of this step shouldbe similar to that obtained from averages across many boutonswhere �r�4 s (4, 16). We reasoned, therefore, that averaging thedata over a timescale corresponding to �r/2 would improve thesignal-to-noise ratio without significant loss of information. Inspec-tion of events filtered in this manner (Fig. 5A) showed a broad rangeof retrieval times that varied among (i) prompt retrieval, i.e., eventswhose decay appears to be dictated solely by reacidification with at1/2 �2.7 s (the t1/2 of a process whose time constant is 4 s) andtherefore whose endocytosis occurred after exocytosis within thetime resolution of the measurement, (ii) events that showed ameasurable delay before the fluorescence decay, and (iii) eventswhose fluorescence failed to decay within the time-measurementwindow (t1/2 � 20 s). We defined the endocytic dwell time ast1/2�2.7. The frequency distribution of endocytic dwell-time valuesfor �150 individual endocytic events indicates these values aredistributed exponentially, well described by a single Poisson processwith a constant transition probability per unit time k that results ina probability distribution P(t) � ke�kt. The mean dwell time � of thedistribution P(t), is equal to 1/k. Here, � � 13.4 � 2.4 s is in closeagreement with the single action-potential ensemble average value(Fig. 2C). Furthermore, the appearance of discrete dwell timesbefore an exponential decay is also evident in ensemble averagetraces of many events drawn from individual times bins (Fig. 5C).These data indicate that, although the mean time for vesicleretrieval after exocytosis is �14 s, individual retrieval events clearlyoccur over a very wide range of timescales. The simple exponentialdistribution of dwell times, however, strongly implies they allrepresent a single stochastic process.

DiscussionWe report here a significant improvement in the ability to monitorexocytosis and endocytosis at the level of single vesicles at individualpresynaptic boutons. The major technical advance was the combi-nation of: (i) a pHluorin-tagged synaptic vesicle protein with verylow surface fraction (�2%) and (ii) high-collection efficiencyoptical readout, capable of single-molecule detection. These im-provements allowed access to quantitative details of exocytosis andendocytosis that permitted us to estimate the time constant forendocytosis at nerve terminals in two ways (Figs. 2C and 5B). Thefirst was a measure of the ensemble response to single AP stimu-lation across many boutons and an examination of the poststimulusdecay. This decay was completely blocked (Fig. 2D) when vesiclereacidification was inhibited, indicating that the normal signal decayarises from endocytosis followed by vesicle reacidification. Our data

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Fig. 4. Quantal size is invariant across different release probability. (A) Noisedistributions (Top) obtained in the absence of stimulation. The solid line is aGaussian fit (�red

2 � 0.6). Middle and Bottom show quantal histograms (bars)obtained by measuring the �F1AP with 1.5 (224 events) and 4 mM (186 events)external calcium, respectively, taken from the same ensemble data as in Fig. 3.The distribution is fit [solid line, �red

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multiple Gaussians (dashed lines are the individual Gaussian components). TheCV for the first nonzero peaks is 0.38 (1.5 mM) and 0.40 (4 mM). (B) Comparisonof quantal size determined from 1.5- and 4-mM histogram, and histogramof all events (Fig. 3B) shows that the quantal size is invariant. (C) Illustra-tive example of time traces from single boutons that we classify as no re-sponse; one quantum and two quanta are shown in Bottom, Middle, and Top,respectively.

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show that the average endocytosis time constant for unitary re-sponses is �15 s, in good agreement with previous estimates (4).The second approach examined the stochastic behavior of decaysignals for individual vesicles and the endocytic dwell-time histo-gram for single-vesicle retrieval. This approach required successfulidentification of events corresponding to single vesicles. Analysis ofthe size distribution of exocytosis signals (Figs. 3 and 4) indicatesthat fluorescent signals corresponding to exocytosis appear quan-tized, and that each vesicle likely contains a single copy of vGpH.The fluorescence decay of individual vesicles reveals two distinctcomponents, a stochastically determined dwell time of residence onthe surface with a mean of �14 s, followed by an exponentialdecline we attribute to reacidification (Fig. 5).

The kinetics of endocytosis at nerve terminals has been a subjectof considerable debate in the last 35 years. Central to the discussionhas been the proposal that a fast mechanism of vesicle retrieval,

kiss-and-run, might operate under certain conditions to enhancesynaptic performance. The evidence for kiss-and-run at small nerveterminals has largely been based on either the observation ofincomplete destaining of organic tracers during stimulation (8, 9,21) or the differential access of fluorescence quenchers comparedwith loss of fluorescent tracers during repetitive cycles of exoendocytosis (10). Here, using methods that allow direct monitoringof fusion, we find that endocytic retrieval is dominated by a singlemode of endocytosis whose average time constant is �14 s.Furthermore, the stochasticity indicates that endocytosis kinetics islikely dominated by a single rate-determining step. It is possible thatprevious evidence of fast endocytosis simply represented a selectivesampling of this distribution due to technical constraints, ascribingdifferent timescales of this stochastic process to different modes ofendocytosis. It is important to note, for example, that for anyPoisson process, although the mean retrieval time is 1/k, the mostprobable retrieval time is at short time intervals.

Recent experiments using siRNA directed against clathrin indi-cated that endocytic recovery in response to single AP requiresclathrin (4) and, by extension, this suggests that the single mode wedescribe here depends on clathrin function. These measurements,however, required averaging over many trials and many boutonsand included a significant correction for photobleaching. Previousmeasurements using pHluorin-based assays with VAMP-2 as thecarrier protein after photobleaching the surface fraction reportedthe three modes of endocytosis (21); however, some of these datahave been questioned as suffering from too low a signal-to-noiseratio to be conclusive (4), and we and others recently showed thatsurface VAMP-2 molecules exchange with vesicular VAMP duringexoendocytosis (15, 22). This, together with photobleaching duringdata acquisition, complicates the analysis and interpretation ofthose results. Electrical capacitance measurements of fusion eventsfrom small synaptic vesicles were recently successfully recorded ina giant calyceal synapse, where flicker-type retrieval with times �1s occurs in �20% of fusion events. It is unclear, however, whetherthese represent one bin of an exponential distribution of a longertimescale process. In our experiments, the fastest events (2.75-sectime bin) represented �17% of all events; however, they weresimply part of a continuous exponential distribution with a meanretrieval timescale of 14 s. If a separate fast mode were present inour experiments, it would manifest as excess events in the first timebin, which were not observed. Our stochastic distribution is welldescribed by a single mean lifetime of �14 s. Although it is possiblethat kiss-and-run might operate only under higher-frequency stim-ulation, endocytosis in this regime was recently shown to dependcompletely on dynamin 1 (3). Although it is premature to assumethat within this single stochastic distribution all events are based onidentical molecular mechanisms, direct tests of the molecular basisof endocytosis should be possible at much higher sensitivity thanwas previously feasible. However, the data on clathrin knockdowns,together with the high-sensitivity measurements presented here, aswell as the analysis of endocytosis during high-frequency activity inthe absence of dynamin 1, all indicate that kiss-and-run must occuronly rarely at these small CNS synapses. Instead, we propose thatthe previous evidence for faster retrieval of synaptic vesicles wassimply one end of an exponential distribution of retrieval times thatare part of a single mode of endocytosis.

In summary, we described a quantitative characterization ofendocytosis at the single-vesicle level indicating that a single modeof endocytosis controls synaptic vesicle retrieval. The stochasticnature of the coupling suggests that a single rate-limiting stepdominates the retrieval time for endocytosis.

Materials and MethodsCell Culture. Hippocampal CA3–CA1 regions were dissected from Sprague–Dawley rat pups, dissociated, and plated onto polyornithine-coated glass cover-slips, as described (23). Optical recordings were performed 14–21 days afterplating, 8–14 days after transfection with vGpH, on coverslips using a laminar-

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Fig. 5. Single-vesicle endocytic dwell time follows an exponential distribu-tion with a mean lifetime of �14 s. (A) Fluorescence traces obtained atindividual boutons after single-vesicle exocytosis. Gray lines are raw timetraces, and the black line is the running average over 13 points (�2 s). Theendocytic dwell time is defined as tdwell � t1/2 ��r ln(2), where t1/2 is the time todecay to 50% of the peak amplitude, and �r is the reacidification time constant(4 s). (B) Frequency distribution of dwell times obtained from 150 single-vesicleevents obtained from four neurons. Bars are the fraction of events decayed inthat time bin, and error bar is the Poissonian noise estimated from the numberof events in that bin. All events that did not decay within the observed time(such as the lower trace in A) are collected in the final bin (not shown) andincluded for calculating the fraction of events. The solid line is a fit to a Poissondistribution. The time constant for a Poisson distribution can be estimatedfrom the exponential and gives 13.4 � 2.4 s as well as reciprocal of theamplitude of the exponential divided by the bin width, which gives 15.0 �1.5 s. The fraction of events that remain undecayed within the observationtime is in good agreement with that obtained by integrating the exponentialfit (17.3%). (C) Average of five events from bins centered at 0.1, 2.85, and 8.35 salong with average of five events that did not decay during the observationtime window is shown. The solid-gray exponential decays are fits to thereacidification time course. The average time constant from the fit is 3.8 �1.8 s.

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flow perfusion and stimulation chamber (volume, �75 �l) on the stage of acustom-built laser-illuminated epifluorescence microscope. Images (512 � 512)were acquired every 170 msec using 100-msec integration with an Andor iXon(model no. DU-897E-BV) back-illuminated EMCCD camera using �1-�W/�m2

488-nm Ar ion acoustooptically shuttered laser whose beam was expanded tofill the field of view, a 40 � 1.3-N.A. Fluor Zeiss objective, and a 1.6 � tube lensusing 515- to 560-nm emission and 510-nm dichroic filters. Action potentials wereevoked by passing 1-ms current pulses, yielding fields of �10-V/cm via platinum-iridium electrodes. Cells were continuously perfused (1–1.5 ml/min, �25°C) in asaline solution containing 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2,25 mM Hepes (buffered to pH 7.4), 30 mM glucose, 10 �M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Research Biochemicals), and 50 �M D,L-2-amino-5-phosphonovaleric acid (AP5; Research Biochemicals). When different[CaCl2] was used, [MgCl2] was increased to keep the divalent ion concentrationconstant. Bafilomycin A1 (Baf, Calbiochem) was used at 0.5 �M and applied for30 s before the beginning of data acquisition. Unless otherwise noted, all chem-icals were obtained from Sigma.

EGFP Studies. EGFP (Biovision) was immobilized in acrylamide gel according toref. 24. Images of several regions in the gel were acquired before and afterbleaching with 100-W Hg Arc lamp illumination for 5 min. Average pixel inten-sities of diffraction limited spots were measured after 3 � 3 smoothing and fieldflattening based on images of a dilute fluorescein solution. Only puncta whoseintensity was �2.5 times the standard deviation of the background signal in theabsence of GFP were used for analysis.

Photobleaching Estimates. Photobleaching rates were estimated by measuringthefluorescencedecayofvGlut1-pHLuorin-expressingboutons inthepresenceofNH4Cl. The bleaching rate for pHluorin in the alkaline state for the majority of thedata was �0.01% per frame. In the acid-quenched state, the bleaching rateshould be �25 times lower.

Image and Data Analysis. Images were analyzed in ImageJ (http://rsb.info.nih.gov/ij) by using custom-written plugins (http://rsb.info.nih.gov/ij/plugins/time-series.html). All visible varicosities were selected for analysis by testing theirresponsiveness to multiple (three to four) rounds of test stimuli delivered as six APtrains at 33 Hz. Only boutons showing fluorescence changes of at least 1quantuminthe16–24stimuliwere includedforfurtheranalysis.Thiseffectivelysetsa lowerbound on the release probability included in the data set of P � 0.06. Averagefluorescence intensities were obtained over a circular region of interest of1.25-�m radius for each bouton that avoided fluorescence decay due to fasterdiffusive processes (4). �F1AP was calculated as two point difference (�Fi) inintensity of the frame (n) on which the stimuli arrived and (n 1) or (n 2),

whichever is greater. A histogram of �F1AP values was fit to multiple Gaussians.For single-vesicle fluorescence decays, the t1/2 time was identified as the firstinstant in time at which the adjacent average has decayed to half the initialamplitude and remains below the 50% intensity point for the remainder of thetrace. The ensemble average curve in Fig. 2C was fit to the function

Ft� � A1e�t�n

� A2e�t�r

� F0,

that describes the time course of two coupled first-order processes with timeconstants for endocytosis (�n) and reacidification [�r, set to 4 s, as determined (4,16)] where amplitudes A1 and A2 are positive vlaues.

Fitting. Levenberg–Marquardt �2 minimization for nonlinear least-square fittingwas carried out in Origin (OriginLab). The areas and positions of each peak thequantal histograms were allowed to vary freely, but the width of all peaks wastaken as a single-fitting parameter, which assumes that the noise in our system isdominated by a combination of systematic instrumentation noise (e.g., drifts,EMCCD clock noise, laser-illumination noise), and the noise associated with abaseline fluorescence derived from the population of acidic vesicles whose flu-orescence is each �1/25th of an alkaline vesicle. We verified the robustness of thequantal description of the data by fitting the derived best-fit peak positions as alinear function of peak number (Fig. 1D); the slope of this fit gives the quantalsize. Quantal-size estimates remained invariant over a wide range of bin sizes, asshown in Table 1. Poissonian error is assumed for calculating the reduced �2 valuein each of our fittings.

ACKNOWLEDGMENTS. We thank Robert Edwards and Susan Voglmaier (Uni-versity of California, San Francisco) for kindly providing the vGlut1-pHluorinconstruct; Ricky Kwan for excellent technical assistance; and Fred Maxfield,Tim McGraw, and members of the T.A.R. laboratory for careful reading of themanuscript. This work was supported by grants from the National Institutes ofHealth (to T.A.R.).

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Table 1. Fitting errors as a function and �2 estimates forquantal analysis

Bin size, a.u. Quantal size, a.u. Error Reduced �2

4.75 52.0 2.0 0.739.75 50.5 2.0 0.97

12.5 49.5 0.9 1.14

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