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The Journal of Neuroscience, March 1, 2002, 22(5):1608-1617
Fast Vesicle Recycling Supports Neurotransmission during
Sustained Stimulation at Hippocampal Synapses
Yildirim
Sara,
Marina G.
Mozhayeva,
Xinran
Liu, and
Ege
T.
Kavalali
Center for Basic Neuroscience and Department of Physiology,
University of Texas Southwestern Medical Center, Dallas, Texas
75390-9111
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ABSTRACT |
High-frequency induced short-term synaptic depression is a common
feature of central synapses in which synaptic responses rapidly
decrease to a sustained level. A limitation in the availability of
release-ready vesicles is thought to be a major factor underlying this
phenomenon. Here, we studied the kinetics of vesicle reavailability and
reuse during synaptic depression at hippocampal synapses. High-intensity stimulation of neurotransmitter release was induced by
hyperosmolarity, high potassium, or action potential firing at 30 Hz to
produce synaptic depression. Under these conditions, synaptic
transmission rapidly depressed to a plateau level that was typically
10-40% of the initial response and persisted at this level for at
least 5 min regardless of the developmental stage of synapses. This
nondeclining phase of transmission was partly sustained by fast
recycling and reuse of synaptic vesicles even after minutes of
stimulation. Simultaneous electrical recording of postsynaptic
responses and styryl dye destaining showed that after an initial round
of exocytosis, vesicles were available for reuse with a delay between 1 and 3 sec during 30 Hz action potential or hypertonicity-induced
stimulation. During these stimulation paradigms, there was a limited
mobilization of vesicles from the reserve pool. During 10 Hz
stimulation, however, the extent of vesicle reuse was minimal during
the first 20 sec. These results suggest a role for fast vesicle
recycling as a functional homeostatic mechanism that prevents vesicle
depletion and maintains synaptic responses in the face of intense stimulation.
Key words:
synaptic vesicle recycling; endocytosis; synaptic
depression; FM1-43; patch clamp; hippocampal culture
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INTRODUCTION |
In the CNS, synapses are faced with
significant challenges to sustain neurotransmission over a wide range
of stimulation frequencies. Structural studies and functional analysis
have shown that central synapses have a small number of functional
synaptic vesicles, especially during early stages of maturation after
synaptogenesis (Vaughn, 1989 ; Fiala et al., 1998; Mozhayeva et al.,
2002). Restrictions on synaptic function imposed by the limited number
of vesicles are partly compensated by recycling and reuse of synaptic
vesicles that have undergone exocytosis (Südhof, 2000; Harata et
al., 2001; Wilkinson and Cole, 2001). These limitations in synaptic vesicle supply are revealed during sustained high-frequency
stimulation, in which most synapses exhibit extensive depression,
measured as a marked decrease in the postsynaptic responses to a
plateau level. A current model of this phenomenon suggests that the
early phase of short-term synaptic depression results from depletion of
vesicles in the readily releasable vesicle pool (RRP), presumably corresponding to vesicles juxtaposed to the active zone, coupled with a
decrease in release probability. During the depressed plateau phase,
synapses are thought to release vesicles that transiently populate the
RRP from a reserve pool (Zucker, 1989; Stevens and Tsujimoto, 1995;
Neher, 1998; Wu and Betz, 1998; Regehr and Stevens, 2001). The vesicles
in the reserve pool are anatomically distant from release sites and
become functionally competent for release during stimulation and
replace vesicles in the RRP. The RRP and the reserve pool together make
up the recycling pool of vesicles, which correspond to all vesicles
capable of activity-dependent recycling on stimulation.
In its current state, this linear model does not take into account the
reuse of recycled synaptic vesicles during synaptic depression. This
omission is supported by experiments conducted in neuromuscular
junction and hippocampal synapses that estimated the time required for
recycled synaptic vesicles to mix with the nonreleased population of
vesicles and to rejoin neurotransmission to be between 40 and 90 sec
(Betz and Bewick, 1993; Ryan et al., 1993; Liu and Tsien, 1995). Recent
studies that examine the recovery of synaptic responses in
"pulse-chase" experiments after depletion of RRP by brief
stimulation showed that RRP vesicles were reused within seconds (Pyle
et al., 2000). However, the extent of vesicle reavailability and reuse
under circumstances in which these mechanisms would be most
advantageous, such as during intense stimulation or at early synaptic
development, has not been determined. To determine the respective roles
of fast vesicle reuse and vesicle replenishment from the reserve pool
during synaptic depression, we monitored vesicle reavailability during
depression induced by multiple forms of sustained stimulation at
distinct stages of synaptic development. In particular, we set out to
determine the correlation between synaptic transmission, which
registers fusion events independent of the use history of vesicles, and destaining kinetics of styryl dye FM2-10, a one-time marker of vesicle fusion.
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MATERIALS AND METHODS |
Cell culture. CA3-dentate gyrus regions were
dissected from hippocampi of 1- to 2-d-old Sprague Dawley rats, and
dissociated cultures were prepared according to previously published
protocols (Kavalali et al., 1999a).
Electron microscopy. The cells were fixed for 30 min in 2%
glutaraldehyde buffered with 0.1 M sodium phosphate, pH 7.2 at 4°C. They were rinsed twice in buffer and then incubated in 1% OsO4 for 30 min at room temperature. After
rinsing with distilled water, specimens were stained en bloc with 2%
aqueous uranyl acetate for 15 min, dehydrated in ethanol, and embedded
in poly/bed 812 for 24 hr. Fifty-nanometer sections were poststained
with uranyl acetate and lead citrate and viewed with a Philips CM-12
transmission electron microscope.
Fluorescence imaging. Synaptic boutons were loaded with
FM2-10 (400 µM; Molecular Probes, Eugene, OR) during a 90 sec incubation in the hyperkalemic solution 45 mM
K+/2 mM
Ca2+. This protocol gives maximal
labeling, as judged by comparison with loading with 1200 action
potentials (APs) applied at 10 Hz, which labels 90% of this total
pool. A modified Tyrode solution used in all experiments contained (in
mM): 150 NaCl, 4 KCl, 2 MgCl2, 10 glucose, 10 HEPES, and 2 CaCl2, pH 7.4 (~310
mOsm). A hypertonic solution was prepared by addition of 500 mM sucrose to the Tyrode solution, and
high-K+ solutions contained equimolar
substitution of KCl for NaCl. Field stimulation was applied through
parallel platinum electrodes immersed into the perfusion chamber
delivering 30 mA 1 msec pulses. All staining and washing protocols were
performed with 10 µM CNQX and 50 µM AP-5 to
prevent recurrent activity. During simultaneous optical and
electrophysiological recordings (see Figs. 5-8), transmission blockers
were omitted from the bath. This omission did not give rise to
significant alterations in destaining kinetics compared with previous
work. Images were taken after 10 min washes in dye-free solution in
nominal Ca2+ to minimize spontaneous dye
loss. In all experiments, we selected isolated boutons (~1
µm2) for analysis and avoided apparent
synaptic clusters (Kavalali et al., 1999a). Destaining of hippocampal
terminals with a hypertonic, high-potassium challenge was achieved by
direct perfusion of solutions onto the field of interest by gravity (2 ml/min). In experiments in which FM2-10-containing solution was washed
out within seconds (see Fig. 4), we increased the perfusion rate to 10 ml/min. Adjustment of the hyperosmotic solution flow rate was critical
to prevent alterations in fluid levels and fluorescence values during
rapid solution exchanges. Fluorescence values were not significantly distorted by cell shrinkage during sucrose application. Images were
obtained by a cooled, intensified digital CCD camera (Roper Scientific,
Trenton, NJ) during illumination (1 Hz and 40 msec) at 480 ± 20 nm (505 dichroic longpass and 535 ± 25 bandpass) via an
optical switch (Sutter Instruments, Novato, CA). Images were acquired and analyzed using Axon Imaging Workbench software (Axon Instruments, Union City, CA).
Electrophysiology. Synaptic responses were recorded from
pyramidal cells using a whole-cell configuration of the patch-clamp technique. Data were acquired using an Axopatch 200B amplifier and
Clampex 8.0 software (Axon Instruments). Recordings were filtered at 2 kHz and sampled at 200 µsec. The pipette internal solution included
(in mM): 115 Cs-MeSO3, 10 CsCl, 5 NaCl, 10 HEPES, 0.6 EGTA, 20 TEA-Cl, 4 Mg-ATP, 0.3 Na3GTP,
and 10 lidocaine N-ethyl bromide, pH 7.35 (300 mOsm).
Picospritzer-delivered pulses of hypertonic sucrose (+500 mOsm) were
applied to proximal dendrites. For AP-dependent stimulation, we
used the same technique as in imaging experiments. Error bars denote SEM.
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RESULTS |
Steady-state level of neurotransmission during
prolonged stimulation
Maturation of presynaptic morphology is associated with an
increase in the number of synaptic vesicles and a delayed emergence of
a sizable reserve pool. The delayed emergence of vesicle pool hierarchy
during development would be expected to have a large impact on the
regulation of synaptic responses to sustained high-frequency stimulation (Mozhayeva and Kavalali, 2000). At early stages of maturation, synapses with few vesicles, of which a large fraction is in
a functionally docked state, should manifest significant synaptic
depression during high-frequency stimulation. Mature synapses, on the
other hand, possess a large number of vesicles and a substantial
reserve pool and thus should be able to sustain neurotransmission for
longer periods. To test these predictions, we characterized the time
course and amount of synaptic depression induced by prolonged (>5 min)
applications of hypertonic sucrose (+500 mOsm) or high-potassium
stimulation (90 mM K+/2
mM Ca2+; Fig.
1). The 5 min time frame provides an
ample period for vesicle depletion and use of the total recycling pool,
primarily because this duration is at least three times longer than the
previous estimates of vesicle exchange and mixing between the RRP and
the reserve pool (Murthy and Stevens, 1999; Pyle et al., 2000).
Surprisingly, both immature [6-7 d in vitro (div);
n = 6] and mature (>15 div; n = 6)
synapses responded robustly to these stimulation paradigms and showed a
significant plateau phase of neurotransmitter release after initial
synaptic depression. Although the size of the total recycling pool
dramatically increases between these two stages of synaptic development
(Mozhayeva and Kavalali, 2000), there were no major quantitative
differences in the time course of synaptic depression and the level of
plateau reached. Most surprisingly, in the case of hypertonic sucrose
stimulation, a rapid rise and decline in neurotransmitter release was
followed by a prominent plateau phase of activity at ~20% of the
peak for both immature and mature synapses (Fig.
1A,B). This sucrose-evoked neurotransmitter release
was vesicular in nature, because in experiments at 6 div in which a
small number of synapses are stimulated, we could clearly identify
individual quantal events even after 300 sec of stimulation (data not
shown). The minutes-long enhancement of baseline activity was in
striking contrast to what is generally thought to be a transient effect
of hyperosmotic shock. A transient mechanical action on synaptic
junctions may explain release of docked vesicles as well as
replenishment of recently vacated docking sites from a reserve
pool; however, it is difficult to account for the minutes-long sustained activity without invoking a form of vesicle recycling operating under these conditions.
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Figure 1.
Prolonged high-potassium or hypertonic sucrose
solution application gives rise to sustained neurotransmission
throughout synapse maturation. A, B, During whole-cell
recordings introduction of hypertonic (+500 mOsm) solution initiated
transmitter release with a peak response followed by a prominent
plateau in immature (6-7 div) and mature (14-16 div) cultures
(n = 6 each). Plots show average
normalized current transfer integrated over 1 sec intervals. Example
traces are depicted in the insets.
C, D, K+ (90 mM)
stimulation caused rapid depolarization and synaptic activity
(n = 6). We isolated the amount of synaptic
activity from the nonsynaptic current influx by subtracting the
baseline current after blockade of postsynaptic receptors by
2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline
(NBQX) (10 µM) and AP-5 (50 µM) during a subsequent application of
high-K+ solution with the same duration. This
baseline typically corresponds to the level indicated by the
dashed line in C. The effect of glutamate
receptor inhibitors during plateau is shown in the
inset. All symbols show mean values ± SEM.
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To corroborate the argument that hypertonic sucrose application causes
a form of continuous vesicle recycling, we performed imaging
experiments in which we applied +500 mOsm solution for 5 min to
synapses loaded with FM2-10, a fast departitioning dye, during 90 sec
application of 45 mM K+/2
mM Ca2+ solution. Within 15 sec of sucrose application, we could destain 16.6 ± 0.1% of the
total fluorescence (n = 3; 221 boutons), whereas at the
end of the stimulation, 245 sec later, the destaining reached 35.9 ± 0.4% of the total. On average, 65% of the initial FM2-10 fluorescence still remained within the boutons by the end of 5 min of
sucrose stimulation. Limited destaining achieved by sucrose application
was in contrast to the sustained level of synaptic activity that could
be induced by this stimulation. These observations support the argument
that the action of hypertonic sucrose stimulation is limited to a
subset of vesicles that correspond to the readily releasable pool. We
also repeated the same electrophysiological and optical protocols on
synapses pretreated with BAPTA-AM for 30 sec to buffer intracellular
Ca2+ in addition to using hypertonic
sucrose stimulation in the presence of EGTA (1 mM; n = 5). Neither the extent of
FM2-10 destaining nor the peak and plateau phase of neurotransmission
induced by sucrose was altered under these conditions, suggesting a
form of vesicle recycling operating independent of intracellular
Ca2+.
Ca2+-dependent high-potassium stimulation
(90 mM K+/2 mM
Ca2+) typically destains up to 60% of the
total fluorescence within 15 sec by mobilizing a larger fraction of the
reserve pool (Klingauf et al., 1998). This form of stimulation evoked
an electrical response pattern similar to hypertonic stimulation,
although the plateau level of transmission reached after the initial
decline was ~10% of the peak regardless of the maturity of synapses
(Fig. 1C,D; n = 6 each). In experiments
using 90 mM K+
stimulation, accurate determination of the synaptic responses required
subtraction of the inward current induced by high-potassium perfusion.
This was achieved by measuring the inward current during 90 mM K+ application in
the presence of postsynaptic glutamate receptor blockers (Fig.
1C, inset). In contrast, electrical responses
induced by sucrose application could be fully blocked by receptor
inhibitors (data not shown).
In a subset of experiments, we induced neurotransmitter release with
extracellular stimulation to trigger action potentials at a frequency
of 30 Hz (Fig. 2). The time constant of
synaptic depression was significantly slower in mature synapses (>10
div; n = 8) compared with synapses at 6-7 div cultures
(n = 9). In the case of mature synapses, responses
declined with a time constant of 4.7 sec, whereas immature synapses
showed a rapid depression with a time constant of 2.7 sec. This
developmental slowdown in the rate of depression is presumably
attributable to a decrease in release probability that was observed
previously in other experimental settings (Bolshakov and Siegelbaum,
1995; Choi and Lovinger, 1997; Pouzat and Hestrin, 1997). Despite
differences in the rate at which the plateau level of transmission is
reached between immature and mature synapses, the relative size of the
sustained transmission at 30 sec after the onset of stimulation was not
significantly different (~25% for each case). Taken together, these
observations indicate the persistence of a substantial level of
synaptic transmission under intense sustained stimulation.
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Figure 2.
Time course of synaptic responses to sustained 30 Hz stimulation. The synaptic activity evoked by the 30 Hz field
stimulation (30 sec) was monitored in immature (top;
n = 9) and mature (bottom;
n = 8) cultures. Young synapses displayed faster
depression kinetics in the first 2 sec of stimulation compared with the
mature synapses. *Statistical difference at p < 0.05. Each bar of the graph represents
total current integrated over 1 sec intervals of field stimulation and
normalized with respect to the maximum. Insets, Evoked
synaptic currents by the first and last 14 pulses during 30 sec
application of 30 Hz stimulation.
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Morphological correlates of vesicle depletion in response to 90 mM K+/2 mM
Ca2+ stimulation
To determine the extent of physical synaptic vesicle depletion
that can be induced with prolonged intense stimulation, we analyzed
electron micrographs obtained from cultures at 8 div stimulated with 90 mM K+/2 mM
Ca2+ stimulation for 10 min and
immediately fixed with glutaraldehyde (Fig.
3). The choice of this paradigm was
prompted by our observation that among the stimulation protocols we
tested previously, 90 mM K+/2
mM Ca2+ resulted in the most
substantial synaptic depression. When compared with control samples
incubated in 4 mM K+/2
mM Ca2+ for 10 min,
high-potassium stimulation resulted in a modest decrease in
the total number of synaptic vesicles (control, 24.4 ± 20.3; n = 63 boutons; after 90 mM K+, 15.8 ± 14.4; n = 87 boutons; mean ± SD). We also
observed several membranous structures presumably of endocytic or
endosomal origin in 90 mM
K+-treated samples (Takei et al.,
1996; Richards et al., 2000) (Fig. 3B). In the electron
micrographs studied, a significant number of synapses contained
synaptic vesicle clusters located near the plasma membrane, although
they did not appear to possess docked vesicles. After 90 mM K+ treatment,
there was a small reduction in the number of these synapses (38% in
control vs 33% after 90 mM
K+). In other boutons, which had
morphologically docked vesicles, there was some decrease in the number
of docked vesicles (control, 4.4 ± 1.5 vs 3.2 ± 1.5 after
90 mM K+).
Relatively moderate alterations in the organization of vesicles in
synapses after intense stimulation argues for a significant role of
vesicle recycling to retain vesicle reavailability and structural
homeostasis of synapses.
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Figure 3.
Prolonged high-K+ stimulation
caused minimal changes in the numbers of vesicles and synapse
morphology. A, B, Example electronmicrographs illustrate
the vesicle organization of synapses from cultures treated with 4 mM K+ (A) and 90 mM K+ (B) for 10 min. Right panels, Cumulative data plotted as the number
of morphologically docked vesicles versus the total number of vesicles
in synapses treated with 4 mM K+
(n = 63 boutons) or 90 mM
K+ for 10 min (n = 87 boutons).
The arrow in B points to one of the
endosomal structures that were abundant after prolonged 90 mM K+ stimulation.
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Synapses retain their fast recycling capability after prolonged
intense stimulation
The relative structural integrity of synaptic boutons after
prolonged 90 mM K+
stimulation, with minimal loss of synaptic vesicles, led us to postulate the involvement of a vesicle-recycling mechanism that remains
intact in fatigued synapses during intense stimulation. To determine
the time course of this recycling process, we performed experiments in
which we first challenged synapses with 5 min of uninterrupted 90 mM K+ stimulation and perfused
the styryl dye FM2-10 (400 µM) for the last 10 sec,
followed by fast washout of the extracellular dye and 90 mM
K+ solution (Fig.
4A). The choice of
FM2-10 was prompted by its fast membrane dissociation kinetics that
results in minimal dye retention in vesicles or membrane infoldings
that form after intense stimulation (Klingauf et al., 1998; Richards et
al., 2000). Destaining induced after a delay of 5 min was used to
assess the extent of loading achieved during this brief application of
FM2-10. At the end of each experiment, we determined the size of the
total recycling pool by measuring maximal dye loading and destaining.
By comparing the two loading levels, we could determine that 10 sec
application of FM2-10 stained ~15% of the total recycling pool (Fig.
4B,C; n = 5; 271 boutons).
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Figure 4.
Time course of vesicle recycling during
sustained high-K+ stimulation. A,
Synapses treated with 90 mM K+ for 5 min
were loaded with FM 2-10 during the last 10 sec of this challenge. The
extent of loading achieved by this protocol was determined by measuring
the amount of dye unloaded with repeated 90 mM
K+ application after 5 min of washout
(n = 5; 271 boutons). To determine the time course
of vesicle reavailability, a brief (10 sec) 90 mM
K+ challenge was interspersed after a delay
( t) after dye loading. In three different sets of
experiments, a 90 mM K+ test was applied
with a delay of 5 sec (n = 7; 368 boutons), 15 sec
(n = 4; 271 boutons), or 30 sec
(n = 4; 213 boutons), respectively. At the end of
the washout period, each trial was evaluated for the remaining FM2-10
staining. After each experiment, we executed a maximal dye loading and
unloading paradigm to determine the total pool size and normalize the
data according to this value. B, Histogram of
fluorescence intensity distributions in fatigued synapses labeled with
the 10 sec staining protocol and total pool size as determined by
maximal FM2-10 staining in controls. C, After 90 mM K+ application for 5 min, up to 15%
of total pool could be stained with FM2-10
(Control). Brief interspersed
high-K+ challenges partially unloaded the synapses.
This fluorescence decrease was first detected at 5 sec after
loading and decreased further with increasing delay indicating that
more recycled vesicles became available for release.
Inset, Percentage of vesicles reavailable with respect
to the delay after initial dye uptake (i.e., the difference between
fluorescence detected after 5, 15, and 30 sec stimulations and
control).
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To estimate the time required for reavailability of vesicles
endocytosed in the last 10 sec of prolonged 90 mM
K+ stimulation, we reapplied 90 mM K+ solution for 10 sec with
a delay of 5, 15, and 30 sec after rapid washout of FM2-10 (Fig.
4A). If a fraction of the stained vesicles was
available for exocytosis during this period, then it should release the
dye in response to this 10 sec 90 mM
K+ stimulation. Furthermore, the extent of
destaining achieved by this interspersed stimulation should increase as
vesicles become reavailable. More than 30% of the vesicles that were
stained with FM2-10 could be unloaded with a second pulse of 90 mM K+ after a 5 sec
delay (Fig. 4C; n = 7; 368 boutons). When
the interval was increased to 15 sec (n = 4; 271 boutons) and 30 sec (n = 4; 213 boutons), the fraction
of vesicles reavailable was saturated at ~50%. This indicates that
half of the endocytosed vesicles were recycled through a slower route
(Fig. 4C).
The interpretation of these experiments heavily relies on the
efficiency of the fast washout of extracellular dye after the initial
loading step. Washout was achieved by a fast perfusion system (10 ml/min), and because of the fast membrane-departitioning time constant
of FM2-10 (0.6 sec; Klingauf et al., 1998), extracellular dye
concentration decreases substantially within seconds. If some residual
FM2-10 was still present to stain synapses during trials with the 5 sec
delay, this would make our estimation of ~30% reavailability at this
time point an underestimate.
Estimation of the kinetics of vesicle reavailability after onset of
intense stimulation
Results in the previous section show that hippocampal synapses are
capable of vesicle recycling at a fast time scale even after 5 min of
intense high-K+ stimulation. The next set
of experiments was designed to complement this estimate by determining
the earliest time point when exocytosed vesicles become reavailable
after the onset of stimulation. To address this question, we examined
the kinetic difference between the rate of FM dye destaining and the
time course of neurotransmitter release from a set of hippocampal
synapses. The rationale behind these experiments stems from previous
observations that during stimulation, FM2-10 can be cleared out of a
fused vesicle within 1 sec by departitioning into solution (Ryan et
al., 1996; Klingauf et al., 1998; Pyle et al., 2000) or within
milliseconds by lateral diffusion in the neuronal membrane (Zenisek et
al., 2000). Both of these time frames are faster than the rate of
fusion pore closure and endocytic retrieval (Klingauf et al., 1998;
Sankaranarayanan and Ryan, 2001). Therefore, recycled vesicles would
not contain significant amounts of FM2-10 that could be detected as
additional destaining, whereas the same vesicles would be refilled with
neurotransmitter after endocytosis that could give rise to additional
synaptic responses. This difference between the two reporters of
synaptic vesicle mobilization should result in a deviation between the kinetics of FM2-10 destaining and neurotransmitter release at a point
at which recycled vesicles start to be reused.
Figure 5 illustrates the procedure we
used to analyze these experiments. In this particular experiment, we
evoked neurotransmitter release by application of hypertonic sucrose
solution onto a pyramidal cell (Fig. 5A). In the same
region, we simultaneously recorded fluorescent destaining kinetics from
synapses that were formed on the dendrites of the same cell (Fig.
5B). Although we have observed a reasonable correlation
between the number of visually selected synapses and the size of
electrical synaptic responses, we cannot exclude potential minor
disparities between the two populations of synapses. Hypertonic sucrose
application was sustained for at least 25 sec until destaining reached
a plateau. In mature synapses, this level corresponded to 30% of the
total pool as determined by multiple applications of
high-K+ stimulation after a 1 min rest
period (Pyle et al., 2000). Destaining profiles originating from all
boutons were averaged and smoothed by fitting with multiple exponential
functions (typically four). The derivative of the smoothed destaining
profile was calculated to obtain the time-dependent change in the rate
of destaining (Fig. 5C,D, dF/dt plot). A
smoothing operation was necessitated by the fact that straight
differentiation substantially increased signal noise and made
comparison with the current signal difficult.
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Figure 5.
Evaluation of sucrose-induced release by
simultaneous electrical recordings and fluorescence destaining.
A, B, Whole-cell electrical recording of a sucrose
response (A) and its instantaneous fluorescence
counterpart from multiple boutons on the same neuron
(B). C, Average fluorescence
response (F, gray line) was smoothed by curve fitting
(dashed line) to reduce noise, which in turn helped
obtain a smooth derivative of the fluorescence signal (dF/dt,
solid line). D, The difference between the rate
of dye release and synaptic activity was assessed after alignment of
the dF/dt and Current plots with respect
to their peaks. Current plot was obtained by integrating
current within 1 sec intervals. The difference shown in the
bottom graph was interpreted as the time course of
vesicle reuse.
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To be able to correlate electrophysiological data at
the same resolution with the rate of FM destaining acquired at 1 Hz, we
calculated the total current during synaptic activity over 1 sec
intervals and normalized with respect to the maximum (Fig. 5D, Current plot). Then we aligned the normalized
dF/dt and current plots with respect to the maximum points of
the two curves (Fig. 5D). In all experiments analyzed, there
was significant agreement between the time courses and maxima of the
two curves. Occasional mismatches in alignment were not larger than 1 sec (Fig. 5D). The result of this analysis revealed a marked
divergence between the rate of FM2-10 destaining and neurotransmitter
release. After subtraction of the two curves, the difference was
interpreted as the time course of recycled vesicles to join
neurotransmission (Fig. 5D, bottom panel).
We followed the same procedure to estimate the time course of vesicle
recycling in response to action potentials induced by extracellular
field stimulation.
Conservation of fast recycling during synaptic development
When we applied this analysis to experiments performed on synapses
at distinct stages of maturation, we observed a consistent divergence
between the rate of FM2-10 destaining and neurotransmitter release
(Fig. 6). At 6-7 div, when synapses
initially assemble a readily releasable pool (Mozhayeva et al., 2002),
the difference between the two curves emerged within seconds and
reached the maximal level with a t1/2 of
2-3 sec in response to sucrose stimulation (Fig.
6A). In the case of 30 Hz field stimulation, there
was larger variability in the delay at which the difference between the
dF/dt and current plots became significant. In these experiments, both time courses followed each other for longer periods, and their difference was ~30-40% smaller than in hypertonic sucrose (Fig. 6B). Interestingly, experiments at later stages of
maturation at 8-9 and 14-16 div showed a similar divergence of dF/dt
and current plots, supporting the presence of a fast recycling scheme that enables exocytosed vesicle to rejoin neurotransmission within seconds. The only noticeable tendency during synapse maturation was
observed in 30 Hz experiments in which the delay at which the vesicles
contributed to neurotransmitter release became progressively shorter
with development (Fig. 6C). This result may suggest an increase in the efficiency of
Ca2+-dependent regulation of the
vesicle-recycling machinery during synapse maturation.
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Figure 6.
Conservation of fast vesicle recycling during
synapse maturation. A, B, Comparison of kinetics of dye
loss and electrophysiological recordings show that the plateau level of
neurotransmitter release cannot be accounted for by styryl dye
destaining. This difference between the two modes of measurement was
present throughout synapse maturation in response sucrose
(A) as well as 30 Hz stimulation
(B). C, Average time delay before
the initial appearance of the difference between two signals during
sucrose (closed circles) and 30 Hz stimulation
(open triangles). D, Rise times
(t1/2) of this difference varied
between 2 and 3 sec during in vitro development [30 Hz
stimulation, n = 9 (6-7 div),
n = 9 (8-9 div), n = 8 (14-16
div); sucrose, n = 8 (6-7 div),
n = 8 (8-9 div), n = 9 (14-16
div)]. All symbols denote mean values ± SEM.
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Morphological and functional studies estimate that the readily
releasable pool of hippocampal synapses contain up to 10 vesicles (Schikorski and Stevens, 1997; for review, see Südhof, 2000). In
our experiments, the vesicles released within the first few seconds of
stimulation with 30 Hz or sucrose correspond to the RRP (Rosenmund and
Stevens, 1996; Pyle et al., 2000). The kinetic difference between the
rate of FM2-10 destaining and neurotransmitter release indicates that
most of these vesicles are reavailable within 5 sec
(t1/2 ~ 2-3 sec) after the onset of
stimulation (Fig. 6D). This time frame therefore
suggests a rate of vesicle reuse of up to two vesicles per second,
indicating a contribution of recycled vesicles to neurotransmission
within seconds after the onset of stimulation.
Estimation of the extent of vesicle pool turnover
during stimulation
An alternative to the analysis presented above involves comparison
of the integral of neurotransmitter release to FM2-10 destaining. This
approach was used previously by Betz and Bewick (1993) to examine
vesicle recycling in the frog neuromuscular junction. In contrast to
differentiation, integration does not increase noise of the current
signal; thus it does not require smoothing. Therefore, this operation
avoids potential errors associated with the smoothing and
differentiation of FM2-10 traces. However, one caveat of the
integration method is the difficulty in proper alignment and scaling of
the electrical and optical signals. To circumvent this problem, first
we used the assumption that both electrophysiological events and FM dye
destaining originate from the same pool of vesicles released within 30 sec of stimulation, disregarding the potential contribution of recycled
vesicles. When we aligned the cumulative integral of
electrophysiological responses and FM-destaining patterns from mature
synapses (8-16 div) using this assumption, we detected a paradoxical
mismatch between the two curves, which suggested FM dye destaining in
the absence of glutamate release (Fig.
7A,B, open
circles). Because FM dye-loaded vesicles were resident within the
recycling pool for at least 10 min before stimulation, all of these
vesicles would also be expected to contain neurotransmitter. This
contradiction could be alleviated once we modified the earlier assumption and presumed that the vesicle pool detected by
electrophysiology is larger than the pool detected by FM dye destaining
because of fast vesicle recycling. To incorporate this
assumption into our calculations, we scaled the
fluorescence trace until it was aligned with the integrated current
trace for the first 2 sec, our previous estimate for the delay in
vesicle reavailability. In the case of hypertonic sucrose stimulation,
FM2-10 destaining could account for only 40% of the electrically
detected neurotransmitter release (Fig. 7A, filled
circles). During 30 Hz stimulation, on the other hand, dye release
comprised 80% of the electrical signal (Fig. 7B,
filled circles). This difference may arise from the fact
that 30 Hz stimulation releases up to 60% of the total pool within
first 30 sec, whereas hypertonic sucrose stimulation is typically
restricted to 20-30% of the total recycling pool (Pyle et al., 2000;
Y. Sara and E. T. Kavalali, unpublished observations).
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Figure 7.
Estimation of the extent of vesicle pool turnover
during stimulation. A, B, Comparison of the cumulative
integral of synaptic current (dark line) to kinetics of
fluorescence loss from FM2-10-loaded synapses in mature cultures (8-16
div). Open circles represent average destaining kinetics
scaled with the assumption that both electrophysiological and optical
readout of exocytosis originate from the same pool of vesicles. Note
the apparent mismatch between the curves. When the fluorescence trace
was scaled to fit the first 2 sec of neurotransmitter release, the
resulting curve (closed circles) revealed a significant
difference between the extent of vesicle pool mobilization and
neurotransmitter release. The same analysis was performed for
hypertonic sucrose stimulation (A) and 30 Hz
field stimulation (B). The relative amount of
vesicle reuse is larger during hypertonic sucrose stimulation. However,
it should be noted that 30 Hz stimulation overall mobilizes a larger
percentage of the total pool compared with sucrose.
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Rate of vesicle reuse during 10 Hz stimulation
Evidence from previous studies at hippocampal synapses has
suggested a slower rate of endocytosis during weak stimulation at
frequencies between 1 and 10 Hz. This difference is attributed to the
effect of intracellular Ca2+ on the
regulation of endocytic machinery (Ryan et al., 1996; Klingauf et al.,
1998; Kavalali et al., 1999b; Sankaranarayanan and Ryan, 2001). To test
the impact of a slower endocytic rate on vesicle reavailability, we
measured the rate of FM2-10 destaining and synaptic depression at 10 Hz
stimulation (n = 10 experiments; 431 boutons total).
During 90 sec of stimulation, fluorescence decreased to 20% of the
initial level (Fig.
8A). In contrast, during 30 Hz stimulation (n = 4; 281 boutons),
fluorescence rapidly decreased to 40% of the initial level, followed
by a slow decline to 30%. Overall, 10 Hz stimulation mobilizes a
significantly larger fraction of the total pool within the first 90 sec. Electrical responses, on the other hand, reach a plateau phase of
~20-30% of the initial peak (Fig. 8B). To
estimate the rate of vesicle reavailability under 10 Hz stimulation, we
applied the analysis described in Figure 5 to synapses stimulated at
this frequency. Alignment of average dF/dt and current plots revealed a
small divergence in kinetics beginning between 5 and 10 sec after onset of stimulation (Fig. 8B). The divergence between the
two curves increased significantly after the initial 20 sec. This
result indicates a slower rate at which exocytosed vesicles are reused for neurotransmission during 10 Hz stimulation, presumably because of
limitations imposed by the rate of endocytic retrieval and vesicle
mobilization at this frequency.
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Figure 8.
Vesicle mobilization during 10 Hz stimulation.
A, In contrast to the biphasic nature of fluorescence
loss during 30 Hz stimulation (n = 4; 288 boutons),
in which a fast drop of fluorescence was accompanied by a slow decline,
10 Hz resulted in monophasic fluorescence loss (n = 6; 431 boutons). The two destaining patterns crossed each other after
50 sec. B, Analysis of average release kinetics acquired
through whole-cell recordings (n = 10) and styryl
dye destaining induced by 10 Hz stimulation. Analysis was performed as
described in Figure 5. The difference between dF/dt and
Current traces became gradually more significant after
20 sec of stimulation. After this point, transmitter release reached to
a plateau level, whereas dye release continued to decrease.
Bottom graph, Difference between the two traces
indicating the time course of vesicle reuse.
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Relationship between the sustained phase of synaptic activity and
kinetics of vesicle reuse
To test whether the kinetic parameters for vesicle reuse
(estimated in the previous section) can account for the sustained phase of synaptic activity during depression, we set up a kinetic model
(Fig. 9A). We restricted this
model to the simplest case of sucrose stimulation in which there was
minimal mobilization of vesicles from the reserve pool as evidenced by
the limited destaining achieved even after prolonged application.
Therefore, initially we placed all vesicles in a single compartment
corresponding to the readily releasable pool
(C0). After sucrose application, vesicles in
compartment C0 could be mobilized to the fused
state C1. We set this rate ( ) at 1/sec,
because this period fairly describes the onset of synaptic responses on
sucrose application. The rate of endocytosis, or the rate of movement
from C1 to C2, on fusion
( ) was set at 1/sec according to previous estimate of Pyle et al.
(2000) under the same stimulation condition. The only variable
parameter was the rate vesicle reuse ( ) through which vesicles
become reavailable for release after endocytosis. When compared with
the average data obtained from prolonged hypertonic sucrose
applications depicted in Figure 1, the initial phase of the plateau up
to 15 sec after onset of stimulation could be well described by vesicle
reuse with a time constant between 5 and 6 sec ( = 0.17/sec),
in close agreement with our estimates from experiments described above
(Fig. 9B). However after this initial phase, the plateau
level declined to ~20% of the peak, which could be accounted for
with a vesicle reuse time constant of 12.5 sec ( = 0.08/sec).
This number is in closer proximity to the time constant of vesicle
reavailability obtained at the end of prolonged high-K+ stimulation, suggesting a gradual
relaxation in the rate of vesicle reuse during sustained stimulation
(Fig. 4C). According to the same model, a vesicle-recycling
time constant of ~100 sec did not result in an appreciable plateau
phase of activity after initial depression, as depicted in Figure
9B. It is interesting to note that this simple kinetic model
can account for our estimates under both sucrose and prolonged
high-K+ application without invoking any
necessity to recruit more vesicles than the ones initially mobilized at
onset. In the case of sucrose, this pool seems to be restricted to the
RRP, whereas high-K+ stimulation mobilizes
a larger population of vesicles comparable with the total recycling
pool.
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Figure 9.
The estimated time frame of vesicle reuse can
account for the sustained phase of synaptic activity. A,
Three-compartmental model where vesicles sequentially move between
compartments C0,
C1, and C2
connected with rate constants , , and , respectively. The
observable output of the model is detected as the fused state
C1 by simultaneous solution of the
equations: dC0/dt =  C0 + C2,
dC1/dt = C0 C1, and
dC2/dt = C1 C2 where = = 1/sec.
B, Correspondence between results of the simulation
described in A (lines) and average data
obtained from prolonged sucrose applications (open
circles). Solid line, Time course of vesicles
moving through the fused state (C1)
when = 0.17/sec ( reuse = 5.8 sec), which
effectively describes the first 15 sec of sucrose response.
Dashed lines, Estimations for distinct values of .
= 0.08/sec can account for the response at the end of sucrose
application. C, Simple scheme depicting the organization
of functionally distinct vesicle pools and the routes of vesicle
replenishment and reuse.
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| |
DISCUSSION |
Contribution of vesicle reuse to neurotransmission during
sustained stimulation
In this study, we evaluated the role of fast vesicle recycling in
sustaining neurotransmitter release during early and late phases of
synaptic depression. These experiments revealed a substantial contribution of recycled vesicles to neurotransmission induced by
hypertonic sucrose, action potentials evoked at 30 Hz and 90 mM K+ stimulation. A
stimulation paradigm as extreme as 90 mM
K+/2 mM
Ca2+ for 5 min could generate
neurotransmitter release continuously with minimal physical loss of
synaptic vesicles. In these experiments, the presence of a sustained
phase of neurotransmission without a significant decline in amplitude
argued for an equilibrium between vesicle reuse and replenishment from
a reserve pool. To determine the contribution of fast vesicle reuse to
this equilibrium, we measured the amount of dye uptake at a point 5 min
after the onset of stimulation. Up to 30% of the vesicles endocytosed
in a 10 sec time frame were reused within 5 sec, supporting a
substantial contribution of vesicle reuse to transmission at this
stage. Overall, up to 50% of the vesicles endocytosed during this
plateau phase were reavailable within 30 sec; the rest of these
vesicles were presumably recycled through a slower pathway.
To quantify the earliest time point at which recycled vesicles are
reused during sustained stimulation, we compared simultaneous optical
measurements of vesicle mobilization and electrical detection of
neurotransmitter release. Destaining kinetics of FM2-10 provides a
one-time marker of exocytosis, because a large fraction (>80%) of the
intravesicular dye leaves the vesicles even during conditions that
favor fast endocytosis (~1 sec), whereas filling of synaptic vesicles
with neurotransmitter can rapidly replenish electrical response after
endocytosis. Thus discrepancies between the two modes of measurement
provide information on the time frame of vesicle recycling during
stimulation. We observed a significant deviation between the derivative
of the average fluorescence destaining and the time course of
electrical responses. The difference emerged at the onset of
stimulation, indicating the presence of a fast recycling process in
which exocytosed vesicles are available for rerelease within 1-3 sec.
Interestingly, the time course of this reuse pathway was conserved
throughout synapse maturation, bringing functional capacity of immature
synapses with small vesicle pools in line with mature presynaptic
boutons (Fig. 9C).
A parsimonious interpretation of these experiments, in terms of vesicle
reuse, depends on the assumption that both optical and electrical
signals are originating from the same population of vesicles. To ensure
the validity of this assumption, we loaded synapses with FM2-10 using
high-potassium stimulation, which gives rise to a maximal amount of
fluorescent labeling in hippocampal synapses (see Materials and
Methods). This loading protocol increases the likelihood that all
recycling vesicles contained dye before their initial fusion. Our
observations may also be explained by delayed release of a vesicle
population followed by a "kiss and run" type of endocytosis on the
order of milliseconds, thus releasing neurotransmitter without dye
destaining (Stevens and Williams, 2000). We consider this possibility
unlikely, because we observed a significant match between the amount of
dye released during 30 sec application of hypertonic sucrose and the
extent of dye loading achieved by the same stimulation (M. Mozhayeva
and E. T. Kavalali, unpublished observations). Styryl dye loading
into synaptic vesicles is less sensitive to fast endocytosis, because these dyes partition into membranes on the order of milliseconds (Neves
and Lagnado, 1999; Pyle et al., 2000).
Among the stimulation paradigms used in this study, the long-term
stability of synaptic responses to hypertonicity was the most
intriguing. It is difficult to reconcile these responses with a purely
mechanical action of osmotic shrinkage (Kashani et al., 2001). This
rather nonphysiological stimulation seems to reveal continuous
recycling capability of an isolated subset of vesicles, presumably the
readily releasable pool, without significant exchange with the reserve
pool in the absence of Ca2+. Elevation in
baseline intracellular Ca2+ levels during
sucrose stimulation did not seem to be involved in this form of
recycling, because experiments conducted after preincubation of cells
in BAPTA-AM to buffer
[Ca2+]i did not
prevent the sustained activity. Previous experiments have shown the
persistence of sucrose-induced neurotransmitter release after extensive
Ca2+ buffering (Rosenmund and Stevens,
1996) but did not address the question of sustained activity during
hyperosmotic stimulation. An interesting corollary of this result is
the presence of a Ca2+-independent form of
endocytosis and vesicle recycling, which may be explained by an
increase in propensity of protein-protein interactions attributable to
a reduction in synaptic volume under hyperosmotic conditions (Delaney
et al., 1991).
Cellular mechanisms that underlie fast vesicle recycling
After exocytosis, synaptic vesicles are recycled and readied for
secondary rounds of release through multiple pathways. A "classical
pathway" involves endocytosis outside the active zone and
incorporates steps to recover fully collapsed vesicles through formation of clathrin coats and recycling through endosomal
intermediates (Heuser and Reese, 1973; Holroyd et al., 1999).
Alternatively, recycling vesicles can form from membrane invaginations
(which may also develop into "cisternae"-like structures) outside
the active zone through clathrin-dependent mechanisms (Takei et al., 1996; Koenig and Ikeda, 1996; Richards et al., 2000). Recent evidence suggests that vesicles may recycle by skipping the endosomal
intermediates and retain their identity through the vesicle cycle
(Koenig and Ikeda, 1996; Murthy and Stevens, 1998; Richards et al.,
2000). Functional evidence in hippocampal synapses also indicates the presence of a more direct pathway where synaptic vesicles can be reused
without ever leaving the active zone, presumably at the original sites
of release (Klingauf et al., 1998; Pyle et al., 2000; Stevens and
Williams, 2000; Südhof, 2000). This pathway is reminiscent of
previous proposals of a kiss and run type of endocytosis by Ceccarelli
et al. (1973). Morphological studies in the Drosophila
neuromuscular junction revealed a ring of endocytic machinery around
active zones. This organization of preassembled machinery would
significantly increase the efficiency and speed of pathways without
endosomal intermediates (Roos and Kelly, 1999). Hot spots for
endocytosis near active zones were also observed in detailed electron
microscopic studies in the snake neuromuscular junction (Teng et al.,
1999; Teng and Wilkinson, 2000). In contrast to previous work,
experiments presented in this study did not rely on the kinetic
difference between destaining rates of synapses loaded with the styryl
dyes FM1-43 and FM2-10. This kinetic difference was interpreted
as an indicator of a kiss and run-like mechanism (Klingauf et al.,
1998; Kavalali et al., 1999b). Therefore, our current results solely
present timing constraints on fast vesicle recycling without any
morphological suggestion for an underlying physical mechanism.
Nevertheless, within the time frames demonstrated in these experiments,
it would be highly unlikely for vesicles to traverse endosomes.
Critical role of fast vesicle recycling as a functional
homeostatic mechanism
In the frog neuromuscular junction, Betz and Bewick (1993)
measured a deviation between the summed amplitudes of endplate potentials and FM1-43 destaining that is detectable at ~90 sec after
stimulation at frequencies between 2 and 30 Hz. This study used an
analysis procedure similar to the one outlined in Figure 7, using the
assumption that neurotransmitter release and FM dye destaining should
line up for the first 30-60 sec of stimulation. Evidently this
assumption may result in an oversight of fast recycling emerging at
early time points (within seconds). Furthermore, in the neuromuscular
junction, FM1-43 labels a large pool of reserve vesicles far exceeding
FM2-10 labeling of a fast recycling pool. This difference may skew
measurements toward slow recycling. This differential labeling of the
two pools with distinct FM dyes may not be as prominent in hippocampal
synapses (Pyle et al., 2000; Harata et al., 2001). However, we also
cannot exclude the possibility that the difference between the results
of Betz and Bewick (1993) and the present study originates from a
genuine difference in vesicle use between the two preparations.
Hippocampal synapses may use faster recycling pathways to ensure more
economical use of their limited vesicle supply. Interestingly, recent
experiments in Drosophila neuromuscular junction that
compared the rate of synaptic depression between wild type and an
endocytosis mutant shibire revealed a recycling rate of one
to two vesicles per second per active zone (Delgado et al., 2000). The
two vesicles per second estimate for fast recycling we provide here is
consistent with these results from the Drosophila
neuromuscular junction.
Taken together, our results support a significant role for vesicle
recycling and reuse in attainment of neurotransmitter release during
sustained stimulation throughout synaptic development. Intense
stimulation patterns with robust onset such as 30 Hz or hypertonic
sucrose activate a rapid pathway where released vesicles are
preferentially reused for neurotransmitter release within seconds.
During 10 Hz stimulation, however, the rate of vesicle reavailability
slows down, and a larger portion of the reserve pool seems to be used
by conventional physical replenishment of the RRP. Therefore, these
results expose the regulation of vesicle-recycling pathways within a
presynaptic terminal as a critical functional homeostatic mechanism
that ensures the most efficient use of the limited number of recycling
vesicles present in a CNS synaptic terminal.
| |
FOOTNOTES |
Received Oct. 24, 2001; revised Dec. 4, 2001; accepted Dec. 13, 2001.
E.T.K is the Effie Marie Cain endowed scholar in biomedical research at
the University of Texas Southwestern Medical Center. We thank Kimberly
Huber, Tuhin Virmani, and Thomas Südhof for helpful discussions
and for critically reading this manuscript.
Correspondence should be addressed to Dr. Ege T. Kavalali, Center for
Basic Neuroscience, University of Texas Southwestern Medical Center,
5323 Harry Hines Boulevard, Dallas, TX 75390-9111. E-mail:
Ege.Kavalali{at}UTSouthwestern.edu.
| |
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Multiple vesicle recycling pathways in central synapses and their impact on neurotransmission
J. Physiol.,
December 15, 2007;
585(3):
669 - 679.
[Abstract]
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G. B. Awatramani, J. D. Boyd, K. R. Delaney, and T. H. Murphy
Effective release rates at single rat Schaffer collateral-CA1 synapses during sustained theta-burst activity revealed by optical imaging
J. Physiol.,
July 15, 2007;
582(2):
583 - 595.
[Abstract]
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J. H. Koenig and K. Ikeda
Release and Recycling of the Readily Releasable Vesicle Population in a Synapse Possessing No Reserve Population
J Neurophysiol,
June 1, 2007;
97(6):
4048 - 4057.
[Abstract]
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M. Ertunc, Y. Sara, C. Chung, D. Atasoy, T. Virmani, and E. T. Kavalali
Fast Synaptic Vesicle Reuse Slows the Rate of Synaptic Depression in the CA1 Region of Hippocampus
J. Neurosci.,
January 10, 2007;
27(2):
341 - 354.
[Abstract]
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A. J. Newton, T. Kirchhausen, and V. N. Murthy
Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis
PNAS,
November 21, 2006;
103(47):
17955 - 17960.
[Abstract]
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A. Menegon, D. Bonanomi, C. Albertinazzi, F. Lotti, G. Ferrari, H.-T. Kao, F. Benfenati, P. Baldelli, and F. Valtorta
Protein Kinase A-Mediated Synapsin I Phosphorylation Is a Central Modulator of Ca2+-Dependent Synaptic Activity.
J. Neurosci.,
November 8, 2006;
26(45):
11670 - 11681.
[Abstract]
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W. J. Tyler, X.-l. Zhang, K. Hartman, J. Winterer, W. Muller, P. K. Stanton, and L. Pozzo-Miller
BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses
J. Physiol.,
August 1, 2006;
574(3):
787 - 803.
[Abstract]
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L. C. R. Tafoya, M. Mameli, T. Miyashita, J. F. Guzowski, C. F. Valenzuela, and M. C. Wilson
Expression and function of SNAP-25 as a universal SNARE component in GABAergic neurons.
J. Neurosci.,
July 26, 2006;
26(30):
7826 - 7838.
[Abstract]
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P. Vanden Berghe and J. Klingauf
Synaptic vesicles in rat hippocampal boutons recycle to different pools in a use-dependent fashion
J. Physiol.,
May 1, 2006;
572(3):
707 - 720.
[Abstract]
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T. Virmani, D. Atasoy, and E. T. Kavalali
Synaptic vesicle recycling adapts to chronic changes in activity.
J. Neurosci.,
February 22, 2006;
26(8):
2197 - 2206.
[Abstract]
[Full Text]
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E. T. Kavalali
Synaptic Vesicle Reuse and Its Implications
Neuroscientist,
February 1, 2006;
12(1):
57 - 66.
[Abstract]
[PDF]
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E. N. Star, A. J. Newton, and V. N. Murthy
Real-time imaging of Rab3a and Rab5a reveals differential roles in presynaptic function
J. Physiol.,
November 15, 2005;
569(1):
103 - 117.
[Abstract]
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T. Takano, J. Kang, J. K. Jaiswal, S. M. Simon, J. H.-C. Lin, Y. Yu, Y. Li, J. Yang, G. Dienel, H. R. Zielke, et al.
Receptor-mediated glutamate release from volume sensitive channels in astrocytes
PNAS,
November 8, 2005;
102(45):
16466 - 16471.
[Abstract]
[Full Text]
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A. C. Ashton and Y. A. Ushkaryov
Properties of Synaptic Vesicle Pools in Mature Central Nerve Terminals
J. Biol. Chem.,
November 4, 2005;
280(44):
37278 - 37288.
[Abstract]
[Full Text]
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J. H. Koenig and K. Ikeda
Relationship of the Reserve Vesicle Population to Synaptic Depression in the Tergotrochanteral and Dorsal Longitudinal Muscles of Drosophila
J Neurophysiol,
September 1, 2005;
94(3):
2111 - 2119.
[Abstract]
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Z. Li, J. Burrone, W. J. Tyler, K. N. Hartman, D. F. Albeanu, and V. N. Murthy
From The Cover: Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin
PNAS,
April 26, 2005;
102(17):
6131 - 6136.
[Abstract]
[Full Text]
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K. L. Moulder and S. Mennerick
Reluctant Vesicles Contribute to the Total Readily Releasable Pool in Glutamatergic Hippocampal Neurons
J. Neurosci.,
April 13, 2005;
25(15):
3842 - 3850.
[Abstract]
[Full Text]
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D. Gitler, Y. Takagishi, J. Feng, Y. Ren, R. M. Rodriguiz, W. C. Wetsel, P. Greengard, and G. J. Augustine
Different Presynaptic Roles of Synapsins at Excitatory and Inhibitory Synapses
J. Neurosci.,
December 15, 2004;
24(50):
11368 - 11380.
[Abstract]
[Full Text]
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Y. Otsu and T. H. Murphy
Optical Postsynaptic Measurement of Vesicle Release Rates for Hippocampal Synapses Undergoing Asynchronous Release during Train Stimulation
J. Neurosci.,
October 13, 2004;
24(41):
9076 - 9086.
[Abstract]
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Y. Otsu, V. Shahrezaei, B. Li, L. A. Raymond, K. R. Delaney, and T. H. Murphy
Competition between Phasic and Asynchronous Release for Recovered Synaptic Vesicles at Developing Hippocampal Autaptic Synapses
J. Neurosci.,
January 14, 2004;
24(2):
420 - 433.
[Abstract]
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N. Axmacher, M. Stemmler, D. Engel, A. Draguhn, and R. Ritz
Transmitter Metabolism as a Mechanism of Synaptic Plasticity: A Modeling Study
J Neurophysiol,
January 1, 2004;
91(1):
25 - 39.
[Abstract]
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S. J Royle and L. Lagnado*
Endocytosis at the synaptic terminal
J. Physiol.,
December 1, 2003;
553(2):
345 - 355.
[Abstract]
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R. P. J. de Lange, A. D. G. de Roos, and J. G. G. Borst
Two Modes of Vesicle Recycling in the Rat Calyx of Held
J. Neurosci.,
November 5, 2003;
23(31):
10164 - 10173.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
