vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses

Hippocampal synapses display a wide range of release

probabilities, release at many synapses being notably

unreliable (Hessler et al. 1993; Rosenmund et al. 1993; Goda

& Südhof, 1997). Precisely which parameters determine

the efficacy of release (the average number of vesicles

released per action potential) at each synapse is unclear,

but the number of vesicles in the readily releasable pool

may be a critical determinant. Furthermore, it has been

suggested that synaptic parameters scale together, a larger

active zone accompanying larger numbers of vesicles in

recycling and readily releasable pools and resulting in a

relatively high release probability (Dobrunz & Stevens,

1997; Murthy et al. 1997; Schikorski & Stevens, 2001). This

‘strict scaling’ model therefore predicts a close relationship

between the number of presynaptic vesicles and synaptic

efficacy.

Most functional studies of hippocampal synaptic properties

have examined release in response to single stimuli or low-

frequency trains (0.5 Hz). Although single unit recordings invivo indicate that CA1 pyramidal cells fire at approximately

1 Hz at rest, during activity (such as running in a wheel)

the firing rate increases to 5–20 Hz and may be sustained

throughout the duration of the activity(Wiener et al. 1989;

Czurkó et al. 1999; Hirase et al. 1999).

Using the FM series of fluorescent styryl dyes (Ryan et al.1993; Betz et al. 1996; Murthy, 1999), we have examined

the release properties of individually identified hippocampal

synapses during stimulation at 1 Hz and at 10 Hz,

corresponding to firing frequencies at rest and during

sustained activity, respectively. Across a large population

of synapses, we observed that different synapses release

different proportions of their recycling vesicle pools per

stimulus during firing at 1 Hz. In contrast, all synapses

released similar proportions during firing at 10 Hz. These

data indicate that, contrary to the strict scaling model,

efficacy scales only loosely with the number of recycling

vesicles at each synapse during firing at low frequencies,

but that the relationship becomes more direct during

higher-frequency stimulation. Furthermore, the degree to

which phorbol ester potentiated release from a synapse

was dependent on the proportion of the recycling vesicle

pool released per stimulus before phorbol application,

suggesting that this coupling might be altered in a synapse-

specific manner by the activation of second messenger

pathways. One explanation of our data is that different

synapses partition different proportions of their recycling

vesicles into the readily releasable pool and that this

parameter therefore influences release probability in a

synapse-specific manner.

Vesicle pool partitioning influences presynaptic diversity andweighting in rat hippocampal synapses Jack Waters and Stephen J Smith

Department of Molecular and Cellular Physiology, Beckman Center, Stanford Medical School, Stanford CA 94305, USA

Hippocampal synapses display a range of release probabilities. This is partially the result of scaling

of release probability with the total number of releasable vesicles at each synapse. We have

compared synaptic release and vesicle pool sizes across a large number of hippocampal synapses

using FM 1–43 and confocal fluorescence microscopy. We found that the relationship between the

number of recycling vesicles at a synapse and its release probability is dependent on firing

frequency. During firing at 10 Hz, the release probability of each synapse is closely related to the

number of recycling vesicles that it contains. In contrast, during firing at 1 Hz, different synapses

turn over their recycling vesicle pools at different rates leading to an indirect relationship between

recycling vesicle pool size and release probability. Hence two synapses may release vesicles at

markedly different rates during low frequency firing, even if they contain similar numbers of

vesicles. Both further kinetic analyses and manipulation of the number of vesicles in the readily

releasable pool using phorbol ester treatment suggested that this imprecise scaling observed during

firing at 1 Hz resulted from synapse-to-synapse differences in the proportion of recycling vesicles

partitioned into the readily releasable pool. Hence differential partitioning between vesicle pools

affects presynaptic weighting in a frequency-dependent manner. Since hippocampal single unit

firing rates shift between 1 Hz and 10 Hz regimes with behavioural state, differential partitioning

may be a mechanism for encoding information in hippocampal circuits.

(Received 4 November 2001; accepted after revision 22 March 2002)

Corresponding author J. Waters: Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29,69120 Heidelberg, Germany. Email: [email protected]

Journal of Physiology (2002), 541.3, pp. 811–823 DOI: 10.1113/jphysiol.2001.013485

© The Physiological Society 2002 www.jphysiol.org

This frequency dependence of the relationship between

recycling vesicle pool size and release probability may shape

the weighting of synapses across a neuronal population in

a firing frequency-dependent manner. This effect may

therefore be an important mechanism, influencing the

output of populations of neurones in a frequency-dependent

manner.

METHODS Preparation of dissociated culturesDissociated hippocampal cultures were prepared from post-natalday 2 Sprague-Dawley rats as previously described (Waters & Smith,2000), in accordance with guidelines laid down by the StanfordUniversity Administrative Panel on Laboratory Animal Care.Pups were killed by decapitation. Hippocampi were dissected andthe dentate gyrus removed. After treating for 15 min at roomtemperature in 10 mg ml_1 trypsin, the tissue was dissociated bytrituration through the tip of a fire-polished siliconized glassPasteur pipette. Dissociated cells were collected by centrifugationat 800 g at 4 °C and plated onto Matrigel-coated coverslips inNeurobasal medium (Life Technologies, Gaithersburg, MD, USA)supplemented with B-27 (Life Technologies), 28 mM glucose,1.3 mM transferrin (Calbiochem, La Jolla, CA, USA), 2 mM

glutamine, 0.7 units ml_1 insulin (Sigma, St Louis, MO, USA) and1 % fetal calf serum (Hyclone, Logan, UT, USA). Cells weremaintained at 37 °C in an atmosphere containing 5 % CO2 untiluse after 10–16 days in vitro.

FM 1–43 staining and destainingA coverslip was mounted in a custom-made, low-volume (60 ml)laminar perfusion chamber on the stage of an inverted micro-scope (Zeiss IM 35). This permitted continuous perfusion atapproximately 1 ml min_1 while imaging through the coverslip towhich the cells adhered. Images were acquired using eithertransmitted light and Nomarski optics or an epifluorescenceconfiguration. Cells were perfused with a modified Tyrodesolution consisting of (mM): 119 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2,25 Hepes and 30 glucose and with 10 mM 6-cyano-7-nitroquin-oxaline-2,3-dione (CNQX), 50 mM D-aminophosphonovalerate(APV) and 3 mM bicuculline added to reduce spontaneous activityand prevent recurrent excitation during stimulation. All imagingexperiments were performed at room temperature (22–23 °C).

Stock solutions of FM 1–43 and FM 2–10 in water were stored at4 °C. FM dyes were diluted into Tyrode solution to a final dyeconcentration of 15 mM and 200 mM, respectively. Followingaddition of FM dye to the perfusing solution, exocytosis wasinduced using a 10 Hz train of 100 stimuli delivered throughplatinum electrodes positioned on opposite sides of the perfusionchamber (stimulus duration 1 ms, field strength 50 V cm_1). Notethat a longer staining protocol was used for the data presented inFig. 2 (see Results). The briefer 100-stimulus protocol was usedfor experiments in which two rounds of FM staining–destainingwere to be performed, since this protocol minimized dye exposureand, therefore, non-specific staining. Control experiments usingthe intracellular calcium dye fluo 4-AM to detect action potential-induced calcium transients verified that stimulation invariablysucceeded in firing neurones at the required frequency.

Following staining, the preparation was washed in dye-freemedium for 10 min to reduce non-specific staining prior to imageacquisition. Subsequent stimulation (at either 1 Hz or 10 Hz)

resulted in destaining of the preparation. Destaining curves werederived from images acquired during this stimulus train.Destaining images were acquired at 3 s intervals during 10 Hzstimulation and at 10 s intervals during 1 Hz stimulation.

All data were adjusted for non-specific staining by subtracting themean fluorescence intensity of each punctum taken from 10images collected following 900 or 1200 destaining stimuli. Thiswas sufficient to release all available vesicles, since furtherstimulation released no additional dye. Although a small decreasein fluorescence was often observed following 1200 stimuli(e.g. Fig. 5A) this was attributable to slight photobleaching of non-specific staining since it also occurred in the absence ofstimulation. It seems unlikely that this gradual photobleaching ofresidual fluorescence could have a strong quantitative influenceon our data since residual staining after 1200 stimuli representedonly approximately 10 % of the fluorescence intensity in thestained condition.

Where data were normalized to the stained condition (see forexample Fig. 2B), data were normalized to the mean fluorescenceintensity from 10 images acquired prior to the destaining stimulustrain. Where a second round of staining–destaining was performed,a 15 min rest period was inserted between the end of the firstdestaining stimulus train and the start of the second stainingstimulus.

Stock solutions of phorbol 12,13-dibutyrate (PDBu) were made at1 mM in DMSO and stored at _20 °C until use. PDBu was added tothe perfusing Tyrode solution to a final concentration of 1 mM.The resulting concentration of DMSO (0.1 %, v/v) did notinfluence FM 1–43 staining or destaining in control experiments.Where used, PDBu was applied for 5 min, beginning 5 min afterthe end of the staining stimulus train and 5 min prior to the startof the destaining stimulus train.

Imaging techniquesThe sample was illuminated using the 488 nm line of an air-cooledargon ion laser at the minimal intensity commensurate withacceptable signal-to-noise of the fluorescent dye (60 mW at theback aperture of the objective). Laser light was focused onto thepreparation using an oil immersion objective lens (w 40, 1.3 NADapo UV, Olympus, Tokyo, Japan) and emitted fluorescence wascollected through an OG 520 nm longpass filter. A Bio-Rad MRC500 confocal laser scanning microscope running dedicated softwarewith custom modifications was used to acquire images. Imageswere stored digitally for off-line analysis using custom software(View, Dr Noam Ziv, Rappaport Institute, Haifa, Israel) or acommercial equivalent (Metamorph, Universal Imaging, WestChester, PA, USA). Fluorescence intensity measurements weretaken from regions of interest of approximately 1.5 mm w 1.5 mm,corresponding to individual puncta, each visibly separate from itsnearest neighbours. Regions of staining larger than 1.5 mm indiameter were excluded from analysis. All fluorescence intensitieswere corrected for non-specific staining by subtracting theintensities measured following complete destaining of thepreparation.

Statistics were performed using commercial software (SigmaStatfor Windows 1.0, Jandel Corporation, San Rafael, CA, USA).Though data were frequently displayed as means ± S.E.M. forpresentation purposes, non-parametric statistics were employedthroughout (see Results). Hence, Wilcoxon’s signed rank test wasused with paired data and the Mann-Whitney rank sum test withunpaired data.

J. Waters and S. J Smith812 J. Physiol. 541.3

RESULTS The FM series of fluorescent dyes has proved extremely

useful as tools for the study of presynaptic vesicle recycling

properties in hippocampal neurones (Ryan et al. 1993;

Betz et al. 1996; Murthy, 1999). We have used FM 1–43

and FM 2–10 and confocal fluorescence microscopy to

monitor vesicle release from hippocampal neurones in

dissociated cultures, such as that shown in Fig. 1A.

After staining with FM 1–43 or FM 2–10 (see Methods)

clusters of presynaptic vesicles were visible as a punctate

fluorescence staining pattern (Fig. 1B). The association of

FM dyes with vesicular membranes is reversible and dye

was released upon subsequent action potential firing

(destaining; Fig. 1C). This procedure could be repeated,

which permitted sequential measurement of presynaptic

properties at the same identified locations since the

position of most fluorescent puncta changed little between

trials (Fig. 1D). Images from the second round of staining–

destaining are included in Fig. 1 (E and F) for comparison

with data from the first round (B and C).

Comparison of destaining at 10 Hz and 1 HzWe used the activity-dependent loss of FM 1–43 staining to

monitor rates of vesicle release in response to stimulation.

We began by staining the preparation using a prolonged

train of 900 stimuli at 10 Hz, leaving FM 1–43 in the

perfusing solution for an additional minute after

termination of the stimulus train. This staining protocol

stains the entire recycling vesicle pool (Ryan & Smith,

1995; Ryan et al. 1996). The fluorescence intensities of

individual puncta were then monitored through time by

acquiring successive images before and during the destaining

stimulus train. Fluorescence intensities recorded from 40

puncta in a single field of view are shown in Fig. 2A. The

fluorescence of each punctum was stable prior to delivery

of the 10 Hz destaining stimulus train. During stimulation,

each punctum destained with an approximately exponential

time course (not shown). Destaining was complete after

Differential partitioning between vesicle poolsJ. Physiol. 541.3 813

Figure 1. Example of sequential FM 1–43 staining and destainingA, Nomarski image of a mixed neuronal–glial culture after 13 days in vitro. B, the same field after stainingwith FM 2–10. The staining protocol consisted of 100 stimuli at 10 Hz then removal of extracellular dye 30 safter cessation of the stimulus train. C, fluorescence image following destaining with a 900 stimulus train at10 Hz. Remaining fluorescence represents non-specific staining, all vesicular staining having been released.D, composite image comparing staining during subsequent trials. Data were corrected for non-specificstaining. Red represents the first round of staining (image C subtracted from image B) and green the secondround (images E _ F), yellow denotes regions where red and green overlap. Note that almost all punctaappear in the same position in the two images indicating that these synapses are positionally stable. E and F,fluorescence images from a second round of staining and destaining using an identical protocol to that forimages B and C. Comparison of E with C reveals an increase in non-specific staining. Note also thatfluorescence intensities are similar following 1st and 2nd rounds of staining. Scale bar represents 10 mm.

900 stimuli. (Note that all data have been corrected for non-

specific staining by subtracting the fluorescence intensity

remaining after 1200 stimuli.)

Release efficacy (measured as loss of fluorescence per

stimulus) was greater for synapses with larger total vesicle

pool sizes (greater initial fluorescence). This is consistent

with published data (Murthy et al. 1997). To examine

whether or not there were substantial differences in the

time constants of release between synapses, we normalized

each trace to its initial fluoresence. Figure 2B illustrates the

resulting ‘destaining curves’ for the 40 fluorescent puncta

represented in Fig. 2A. Only modest differences in the

fraction of initial fluorescence lost per stimulus (fractional

destaining rate) were observed between synaptic puncta

across numerous preparations.

In contrast, in response to a 1 Hz stimulus train, fractional

destaining rates displayed marked heterogeneity across the

synaptic population (Fig. 2C). Some synapses destained so

slowly in response to a 1 Hz train that a subsequent 10 Hz

train was necessary to complete destaining of the preparation

(Fig. 2C). Similar heterogeneity of fractional destaining

rates was observed in numerous preparations in response

to 1 Hz stimulation.

One possible explanation for the heterogeneity observed at

1 Hz might be that failure of action potential initiation

and/or propagation influences release from some synapses

more than others, perhaps due to the relative position of

different synapses with regard to axonal branching patterns.

To address this possibility we monitored destaining at

10 Hz after reducing the extracellular calcium concentration

to 0.1 mM. This should decrease action potential threshold

and favour propagation, but decrease presynaptic calcium

influx and therefore presynaptic efficacy. Pronounced

heterogeneity was observed under these conditions (data

not shown) indicating that heterogeneity is related to

synaptic efficacy, not action potential failure.

We have examined this frequency-dependent heterogeneity

in further detail, repeating rounds of FM dye staining and

destaining to make two measurements of activity at each

synapse. A comparison of destaining curves at 1 Hz and

10 Hz is presented in the form of a frequency histogram in

Fig. 3A. As anticipated from Figs 2B and C, a greater range

was observed at 1 Hz than at 10 Hz. The destaining curves

of rapidly destaining puncta followed an exponential time

course (not shown). However, it was not possible to

reliably fit the decay time course of those puncta which

displayed minimal destaining during the 1 Hz train. We

have therefore expressed fractional release rates as a

percentage of fluorescence released (percentage destaining)

after 150 destaining stimuli.

If the fractional rates observed at 1 Hz reflect differences

between synapses, rather than fluctuations in release or

measurement artifacts, one would expect the rates at each

synapse to be reproducible. Comparison of the fractional

destaining rates at 1 Hz for each synaptic punctum during

sequential trials indicated that fractional destaining rates

at 1 Hz were indeed reproducible (Fig. 3B) and were

therefore characteristic of each synapse. In addition, no

correlation was observed between fractional rates at 1 Hz

and total recycling vesicle pool size (Fig. 3C; n = 333

puncta).

Since small differences in measurement error between

experiments may obscure subtle correlations, we also

examined the relationship between fractional destaining

rate and total pool size in each individual experiment. Data

from one experiment are displayed in the inset in Fig. 3C.

In no single experiment was any relationship between total

pool size and fractional destaining rate at 1 Hz evident. To

address the specific possibility that synapses with a small

total pool size displayed slower fractional release rates at

1 Hz than the population mean (see Discussion), we

compared the mean initial fluorescence of the synapses

with slowest fractional destaining rates with that of the

population of synapses as a whole. For the experiment

displayed in the inset to Fig. 3C, the 10 % of synapses with

the slowest fractional destaining rates had a mean (±S.E.M.)

initial fluorescence of 29.2 ± 3.0 arbitrary fluorescence

units. By comparison, the whole population had a mean

(± S.E.M.) initial fluorescence of 32.5 ± 1.3 arbitrary

fluorescence units. The total pool size of the slowest

destaining synapses was not significantly different from

that of the whole population (P = 0.55, Mann-Whitney

rank sum test).

The influence that these differences in fractional destaining

rates have on synaptic efficacy is illustrated in Fig. 3D, which

shows the number of vesicles (amount of fluorescence)

released in response to 150 stimuli at 1 Hz as a function of

recycling pool size (initial staining intensity). There is a

clear trend for synapses with larger recycling pool sizes to

release more vesicles than those with smaller recycling

pool sizes, consistent with published observations (Dobrunz

& Stevens, 1997; Murthy et al. 1997). However, recycling

pool size is not the only determinant since different

synapses may release different amounts of dye despite

exhibiting similar initial staining intensities.

These observations indicate that any two synapses may

release vesicles at markedly different rates during low-

frequency firing, even if they contain similar numbers of

vesicles. Release rates are reproducible and so are

characteristic of any given synapse. In contrast, hetero-

geneity across the synaptic population is much less

pronounced at 10 Hz.

Heterogeneity at 10 Hz during the initial phase ofreleaseAlthough pronounced heterogeneity was not observed at

10 Hz, small differences in the fractional rates of destaining



Figure 2. Pronounced kinetic heterogeneity at 1 Hz, but not 10 Hz

A, fluorescence intensities of 40 individual fluorescent puncta followed through time. Traces begin in the

stained condition. Staining consisted of 900 stimuli at 10 Hz, followed by an additional minute in FM 1–43

then a 10 min wash period. During stimulation at 10 Hz, each punctum loses fluorescence through time.

Note that all puncta are fully destained by 900 stimuli at 10 Hz. Data were corrected for non-specific staining

by subtracting the fluorescence intensity after 1200 stimuli. B, the same data following normalization to the

fully stained condition. C, similar data derived using the same loading protocol, but using a 1 Hz destaining

stimulus train. Note that some synapses destained only very slowly, if at all, necessitating the use of a 10 Hz

stimulus train (arrow) to complete destaining. Data from the fully destained condition are visible on the

right.

between synapses were observed at this frequency (see

Fig. 2B). To determine whether these small differences in

fractional destaining rates at 10 Hz were related to the

more substantial differences observed at 1 Hz, we

performed two rounds of staining–destaining once at each

frequency (1 Hz and 10 Hz). This permitted a direct

comparison of destaining in response to these two

frequencies at the same, identified synapses. To maximize

the temporal resolution of our measurements, we used

FM 2–10 rather than FM 1–43 (Ryan et al. 1996). To

compare fractional rates at these two frequencies we

ranked 214 puncta according to their fractional rates at


Figure 3. Properties of 1 Hz heterogeneityA, frequency histogram comparing the extent of destaining following 150 stimuli at 1 Hz and 10 Hz.Frequency is displayed as the percentage, rather than absolute number, of puncta in each bin to allow directcomparison of 1 Hz and 10 Hz data, despite different numbers of observations. Data represent 616 puncta at1 Hz and 375 puncta at 10 Hz. B, scatter plot illustrating the 1 Hz fractional destaining rates of 333 punctaduring two subsequent trials. Each point represents the destaining percentages of a single fluorescentpunctum after 150 stimuli at 1 Hz in each trial. The data were best fitted with a regression line of slope 1.009passing through the origin. C, scatter plot comparing 1 Hz fractional destaining rates with initialfluorescence intensity in the stained condition. The staining protocol consisted of 100 stimuli at 10 Hz.Fractional destaining rates are represented as percentage destaining after 150 stimuli at 1 Hz. Small pointsrepresent the values of 333 individual puncta. Large circular symbols represent the mean (± S.E.M.) values forthe same data binned according to their rates of destaining (bins each represent 10 percentage units). Inset,scatter plot comparing 1 Hz fractional destaining rates with fluorescence intensity in the stained conditionfor a single experiment (108 puncta). D, plots showing the absolute amount of fluorescence released inresponse to 150 stimuli at 1 Hz as a function of initial fluorescence staining intensity. Each of the 333 datapoints represents data from one synapse.

1 Hz, then compared the mean fractional rates at 10 Hz of

the 50 fastest and 50 slowest puncta. The data indicate that

puncta that destained slowly and rapidly at 1 Hz also

destained at different fractional rates at 10 Hz (Fig. 4A).

Further examination of the data revealed that the

difference in fractional destaining rates between the two

curves was related to the initial phase of destaining.

Figure 4B shows the two 10 Hz destaining curves from

Fig. 4A presented in a different manner: they have each

been normalized to their respective fluorescence values

attained after 150 destaining stimuli. Clearly, the difference

between these two curves is limited to the initial phase of

destaining. This indicates that fractional destaining rates at

1 Hz are related to the fractional rates during the initial

(but not later) phase at 10 Hz. To determine the precise

duration for which 1 Hz and 10 Hz rates correlate, we

manipulated the data in another fashion, calculating the

percentage destaining occurring between each sequential

pair of images (see legend to Fig. 4B). The resulting data

are presented in Fig. 4C. A significant difference was

observed between the fractional rates of destaining after 30

and after 60 stimuli at 10 Hz, but not thereafter (P < 0.01,

Mann-Whitney rank sum test). These data therefore

indicate that fractional rates at 1 Hz are correlated with

fractional destaining rates during the initial, but not

subsequent, phase of destaining at 10 Hz.


Figure 4. Direct comparison of 1 Hz and 10 Hz destaining curvesA, comparison of the fractional destaining rates at 10 Hz of two groups of puncta stained with FM 2–10: thosewith rapid and those with slow fractional rates at 1 Hz (filled and open circles, respectively). Data pointsrepresent the mean (± S.E.M.) of 50 puncta (of a total population of 214). Inset, mean destaining curves at1 Hz for the same two groups. The asterisk denotes the data point after 150 stimuli (see below). B, the same10 Hz curves (presented in A) normalized to the fluorescence intensity after 150 destaining stimuli. Note thatthe curves converge after the first few images. To analyse this phenomenon in more detail the percentagedestaining occurring between each image pair was calculated for each punctum. For instance, the percentagedestaining occurring between images labelled A and B was calculated using the following formula:% destaining = 100 w [(intensity in A) _ (intensity in B)]/(intensity in A). These data are presented in C.C, mean (± S.E.M.) destaining per image pair. Values were calculated separately for each punctum thenpooled as in A. Asterisks denote a significant difference between destaining rates (P < 0.01, Mann-Whitneyrank sum test). D, scatter plot comparing percentage destaining by 30 stimuli at 10 Hz and total pool size.Data were derived from experiments using FM 1–43. Small points represent 476 individual puncta and largecircular symbols represent mean (± S.E.M.) of the same data grouped into 3-unit bins.

Since no correlation was observed between fractional rates

at 1 Hz and total pool size (see Fig. 3C) these data relating

fractional rates at 1 Hz and at 10 Hz lead to a further

prediction: that the initial rate of destaining at 10 Hz

should not correlate with total pool size. The data

presented in Fig. 4D demonstrate that this is indeed the

case.

Effects of phorbol esterIt has been suggested that initial release probability during

10 Hz stimulation is related to the number of vesicles

contained within a readily releasable pool (Dobrunz &

Stevens, 1997). Furthermore, it has been suggested that

phorbol esters may promote release by increasing the

number of vesicles in the readily releasable pool (Stevens &

Sullivan, 1998; Waters & Smith, 2000). We have therefore

examined the effects of phorbol ester on the heterogeneity

of destaining rates at both 1 Hz and 10 Hz.

To examine the effects of phorbol ester, one round of

staining–destaining was conducted then vesicles were

stained a second time before phorbol application. Phorbol-

12,13-dibutyrate (PDBu; 1 mM) was applied for 5 min

immediately prior to the second round of destaining

(Waters & Smith, 2000). Phorbol treatment resulted in an

increase in fractional release rate at 10 Hz (Fig. 5A). No

change occurred in controls, which were not treated with

PDBu (data not shown). Manipulating the data as before

(see Fig. 4C), it was evident that the effect of PDBu was

limited to the first two data points on the destaining curve

(Fig. 5B).

Since this effect could potentially be influenced by the

slow off-rate of FM 1–43, we repeated these experiments,

using FM 2–10 (Fig. 5C and D). Quantitatively similar

potentiations were observed with both dyes, indicating

that the slower off-rate of FM 1–43 does not substantially


Figure 5. Effect of PDBu at 10 HzA, 10 Hz destaining kinetics measured with FM 1–43 before and after PDBu treatment. Data points representmean (± S.E.M.) measurements from 141 puncta. B, same data represented as percentage destaining betweeneach image, as in Fig. 4C. The effect of PDBu was significant after 30 and 60 destaining stimuli (P < 0.01,Wilcoxon’s signed rank test) where destaining was increased by 110 % and 25 % respectively. C, 10 Hzdestaining kinetics measured with FM 2–10 before and after PDBu treatment. Data points represent mean(± S.E.M.) measurements from 138 puncta. D, percentage destaining between each image for destainingcurves derived using FM 2–10. The effect of PDBu was significant after 30 and 60 destaining stimuli(P < 0.01, Wilcoxon’s signed rank test). The degree of potentiation was also similar to that with FM 1–43,being 115 % and 50 % after 30 and 60 stimuli, respectively.

influence our measurements. Since fractional release rates

at 1 Hz are related to initial rates at 10 Hz, one might also

expect an effect of phorbol esters at 1 Hz. Figure 6Aillustrates such an effect on FM 1–43 destaining. No change

occurred in controls, which were not treated with PDBu

(data not shown).

These data indicate that phorbol ester treatment changes

mean synaptic release properties. It was possible that

phorbol ester alters release properties in a synapse-specific

manner, thereby influencing heterogeneity. We therefore

sought to determine whether or not the initial release rate

of a synapse could predict its sensitivity to PDBu. The

effect of PDBu on release rates at 10 Hz was most

pronounced at the data point taken after 30 stimuli (see

Fig. 5B). We therefore compared release induced by a

30-stimulus train at 10 Hz before and after PDBu

treatment. Fig. 7A shows these data presented to illustrate

the relationship between initial fractional release rate and

the effect of PDBu. Only those synapses which released less

than approximately 20 % of their recycling vesicles were

sensitive to PDBu. After PDBu treatment the percentage of

vesicles released was similar across the population of

synapses.

At 1 Hz, synapses displayed a similar differential sensitivity

to PDBu. Synapses that released more than approximately

20–30 % of their recycling vesicles in response to 30 stimuli

at 1 Hz were insensitive to PDBu (Fig. 7C). As at 10 Hz,

PDBu partially compensated for the lower fractional release

rates of some synapses at 1 Hz (Fig. 7D). Hence PDBu

treatment has an ‘equalizing’ effect on release at both 1 Hz

and 10 Hz; after PDBu treatment, all synapses release an

equal proportion of their vesicle pools in response to a

brief stimulus train.

DISCUSSIONWe have examined the relationship between stimulus

frequency and fractional release rates across a large

number of hippocampal synapses. Striking heterogeneity

was observed upon stimulation at 1 Hz, the population of

synapses displaying a wide range of fractional destaining

rates. Repeated measurements revealed that fractional

destaining rates at 1 Hz were reproducible and therefore

characteristic of each synapse. In contrast, at 10 Hz

heterogeneity was less pronounced and the duration of

heterogeneity was limited to approximately the first 60

stimuli. Similarly, the effect of phorbol ester on destaining

at 10 Hz was limited to the first 60 stimuli.

Previous authors have described the release properties of

hippocampal synapses in terms of the segregation of vesicles

into different pools. Two functional pools have been

proposed. The recycling vesicle pool consists of all vesicles

that will recycle in response to electrical activity. The

readily releasable pool, a subset of the recycling pool,

consists of vesicles which are preferentially released,

possibly because they are already docked at the active zone

and/or primed for release. Correlations have been observed

between (i) release probabilty and the number of vesicles

in the readily releasable pool (Dobrunz & Stevens, 1997),

(ii) the numbers of vesicles in total recycling and readily

releasable pools (Murthy & Stevens, 1999), and (iii) total

recycling pool size and release probability (Murthy et al.1997). In addition, morphological studies have revealed


Figure 6. Effect of PDBu at 1 HzA, 1 Hz destaining curves generated with FM 1–43 beforeand after PDBu treatment. Data points represent mean(± S.E.M.) measurements from 78 puncta. B, same datarepresented as percentage destaining between each image,as in Fig. 4C. The effect of PDBu was significant after 10,20, 30, 40, 50 and 60 destaining stimuli (** P < 0.01,* P < 0.05, Wilcoxon’s signed rank test) where destainingwas increased by 78 %, 46 %, 54 %, 74 %, 78 % and 74 %,respectively.

correlations between other parameters such as number of

docked vesicles and area of the active zone (Schikorski &

Stevens, 1997). One interpretation of these studies is that

many presynaptic properties scale in a stringent manner,

such that two synapses with similar active zone areas will

contain similar numbers of recycling vesicles, have similar

readily releasable pool sizes and so display similar release

probabilities.

Viewed in terms of these multi-pool models, the hetero-

geneity we observed may be related to the readily releasable

pool of vesicles, since (i) the readily releasable pool strongly

influences the efficacy of release in response to single

stimuli and, therefore, during firing at 1 Hz (Dobrunz &

Stevens, 1997), (ii) the readily releasable pool is responsible

for release during only the first few tens of stimuli at 10 Hz

(Dobrunz & Stevens, 1997; Murthy & Stevens, 1999), and

(iii) phorbol esters increase the number of vesicles in the

readily releasable pool (Stevens & Sullivan, 1998). Hence,

different fractional destaining rates would result if either

(i) the release probability per readily releasable vesicle

differs from synapse to synapse, or (ii) the proportion of

recycling vesicles in the readily releasable pool differs from

synapse to synapse. These two explanations are not mutually

exclusive, but each might potentially account for the

observed heterogeneity without recourse to the other.

These two possible explanations are considered below.

Dobrunz & Stevens (1997) found a close correlation between

release probability and readily releasable pool size for single

hippocampal synapses. This suggests that variation in release

probability per readily releasable vesicle is minimal and,

therefore, that the heterogeneity that we observed is unlikely

to reflect different release probabilities per readily releasable

vesicle.


Figure 7. Effect of PDBu depends on initial synaptic propertiesA, plot to show the relationship between the effect of PDBu and the proportion of recycling vesicles in thereadily releasable pool before PDBu treatment. Release was induced by 30 stimuli at 10 Hz. Data from 774puncta were binned according to the percentage released before PDBu treatment (2-unit bins) and areplotted as means (± S.E.M.). B, same data presented to compare release before and after PDBu treatment. Theline indicates the relationship expected if PDBu treatment were excluded. C and D, similar data to thosepresented in A and B, but using a 1 Hz stimulus (30 stimuli). Data represent a total of 417 puncta.

Our alternative hypothesis concerns the proportion of re-

cycling vesicles in the readily releasable pool. As discussed

above, previous authors have found a correlation between

recycling and readily releasable pool sizes, suggesting that

the proportion of vesicles in the readily releasable pool

is constant (Murthy & Stevens, 1999). These previous

conclusions were derived from measurements at individually

identified synapses. However, the conclusions derived

from these studies were derived from mean relationships

between synaptic parameters, measured across populations

of synapses. It is possible, therefore, that these data describe

the mean trends across synaptic populations, but that the

properties of individual synapses may deviate substantially

from these trends.

Our hypothesis is that the proportion of recycling vesicles

in the readily releasable pool differs from synapse to

synapse and that this accounts for the heterogeneity of our

data. This hypothesis may be consistent with published

data if the mean proportion of recycling vesicles in the

readily releasable pool is equal to that of these earlier

studies. The mean fractional destaining observed in our

experiments after 60 stimuli at 10 Hz was 31.9 ± 1.1 %.

This is remarkably similar to the figure reported by

Murthy & Stevens (1999) who calculated that 32 % of

recycling vesicles were within the readily releasable pool.

The mean recycling vesicle pool size was also similar at all

initial fractional destaining rates at 10 Hz (Fig. 4D), again

consistent with the observations of Murthy & Stevens

(1999). In view of the above discussion, it is important to

note that we observed correlations between total recycling

pool and readily releasable pool sizes and release

probability similar to those reported by previous authors

(data not shown).

We therefore consider it likely that heterogeneity in

fractional destaining rates at 1 Hz and during the initial

phase of destaining at 10 Hz reflects synapse-to-synapse

differences in the proportion of vesicles in the readily

releasable pool. Although we cannot categorically exclude

other possible mechanisms, or a combination of two or

more mechanisms, we consider that this hypothesis

provides the most likely explanation for our data.

The effects of phorbol ester are readily explained by this

hypothesis. At both 10 Hz and 1 Hz, phorbol ester treatment

strongly potentiated release only at synapses with low

fractional release rates. The lack of sensitivity of some

synapses to PDBu may indicate that there is a ceiling on the

proportion of the recycling pool which can be maintained

in the readily releasable condition and that this ceiling had

been reached at some synapses prior to PDBu treatment.

Our data indicate that a physiological stimulus acting in a

similar manner to phorbol esters, such as activated protein

kinase C, may push the proportion of recycling vesicles in

the readily releasable pool towards this ceiling value,

altering the frequency dependence of transmitter release in

a synapse-specific manner.

Our conclusions, detailed above, lead to the following

model of synaptic release. The efficacy of release in

response to a single stimulus or during low-frequency

firing (insufficient to induce facilitation or depression) is

determined principally by the number of vesicles in the

readily releasable pool. Dobrunz & Stevens (1997) also

arrived at this conclusion following detailed examination

of the properties of individual synapses using electro-

physiological techniques and the hippocampal slice

preparation. Our data suggest that the size of the readily

releasable pool is determined by two factors: the total

recycling vesicle pool size and the proportion of these

vesicles in the readily releasable condition. As a result,

mean synaptic efficacy scales with total pool size. However,

scaling is imprecise since the release properties of each

individual synapse may be dominated by the proportion of

its recycling vesicles that are partitioned into its readily

releasable pool. The stringency of scaling can therefore be

increased by forcing the proportion of recycling vesicles in

the readily releasable pool towards the same value at all

synapses. This can be achieved in either of two ways: by

exhausting the readily releasable pool using a sustained

high-frequency stimulus (which pushes the proportion in

the readily releasable pool towards zero at all synapses) or

by phorbol ester treatment (which forces the proportion in

the readily releasable pool towards its ceiling value).

This model is also consistent with studies at other CNS

synapses indicating that release probability is not determined

by vesicle pool size alone. At the calyx of Held, for instance,

recycling pool size and release probability change in

opposite directions during development from P5 to P14,

effectively compensating for each other (Taschenberger &

von Gersdorff, 2000; Iwasaki & Takahashi, 2001). Clearly,

these two parameters do not scale at the calyx of Held.

Similarly, the release probabilities at climbing and parallel

fibre synapses in the cerebellum are markedly different

despite similar docked pool sizes (Xu-Freedman et al.2001) indicating that parameters other than docked pool

size must influence release probability at one or both of

these synapses. That pool size is not the only determinant

of release probability has also been suggested for hippo-

campal synapses, where ultrastructural studies have

indicated that the proportion of vesicles released differs

substantially between synapses (Harata et al. 2001).

Although the underlying mechanisms controlling release

probability are not clear at each of these synapses, these

data clearly preclude stringent scaling with vesicle pool

size.

Although our data suggest that vesicle partitioning between

recycling and readily releasable pools is an important

determinant of release probability at hippocampal synapses,


other factors are also likely to be important and may

contribute to the synaptic variability that we have observed.

One such factor is presynaptic receptors, which have been

shown to modulate release at the calyx of Held (Takahashi

et al. 1996), in the locus coeruleus (Dubé & Marshall, 2000)

and mossy fibre synapses in the hippocampus (Scanziani etal. 1997). Another possible variable is the extent to which

release occurs without fusion and mixing of the vesicle and

presynaptic plasma membranes. The latter could permit

staining, but not destaining of synaptic vesicles (Henkel &

Betz, 1995). If release were to occur by two pathways, one

entailing complete fusion of presynaptic vesicles and

another incomplete fusion, the balance between these two

pathways might also contribute to the synaptic variability

that we have observed.

Implicit in our conclusions is the assumption that the

intensity of fluorescence staining is linearly related to the

recycling vesicle pool size. In order for this assumption to

be accurate, all recycling vesicles must be stained with dye

and all stained vesicles must emit equal fluorescence.

Previous authors have shown that staining with just a few

action potentials yields fluorescence intensity distributions

consistent with equal staining of each vesicle (Murthy

et al. 1997; Ryan et al. 1997). Hence our fluorescence

measurements should accurately represent the number of

vesicles stained. Whether all released vesicles are stained is

less clear and we cannot exclude the possibility that some

vesicles recycle without becoming stained with FM dye.

However, our estimates of recycling pool size arise from

FM dye staining using high-frequency stimulation (10 Hz).

FM staining seems to correlate quite well with the size of

the docked vesicle pool following high-frequency stimulation

(Schikorski & Stevens, 2001). Hence, although it is possible

that incomplete FM staining of the recycling pool may lead

to an underestimate of the number of vesicles in the

recycling pool, any such underestimate is likely to be

minimal.

We have shown that, at the level of individual synapses,

synaptic efficacy is less directly related to recycling vesicle

pool size than was previously thought. Hence recycling

vesicle pool size and synaptic efficacy are related by a

frequency-dependent variable, these two parameters being

tightly linked only during sustained firing at high

frequencies, such as 10 Hz. As a result, the relationship

between vesicle pool sizes and synaptic efficacy may

change through time during firing patterns observed invivo (Czurkó et al. 1999; Hirase et al. 1999). Activation of

phorbol ester-sensitive signalling cascade(s) also tightens

the relationship between total pool size and synaptic

efficacy. In this manner, sustained firing and second

messenger cascades each exert synapse-specific influences

on synaptic weighting. This mechanism may therefore

have important implications for the encoding of

information and output of neural circuits.

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Acknowledgements We would like to thank Murali Prakriya and Charles F. Stevens foradvice and comments on the manuscript. This work was supportedby funds from the NIMH Silvio Conte Center for NeuroscienceResearch (MH48108) to S.J S.


vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses

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