vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses
TRANSCRIPT
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
J. Waters and S. J Smith814 J. Physiol. 541.3
Differential partitioning between vesicle poolsJ. Physiol. 541.3 815
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
J. Waters and S. J Smith816 J. Physiol. 541.3
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.
Differential partitioning between vesicle poolsJ. Physiol. 541.3 817
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
J. Waters and S. J Smith818 J. Physiol. 541.3
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
Differential partitioning between vesicle poolsJ. Physiol. 541.3 819
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.
J. Waters and S. J Smith820 J. Physiol. 541.3
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,
Differential partitioning between vesicle poolsJ. Physiol. 541.3 821
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.
Differential partitioning between vesicle poolsJ. Physiol. 541.3 823