SYNAPSE 47:184 –199 (2003)
Presynaptic Quantal Plasticity: Katz’s
Original Hypothesis Revisited
JEAN VAUTRIN* AND JEFFERY L. BARKER
Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke,
NIH, Bethesda, Maryland 20892, USA
KEY WORDS
transmitter release; calcium spike; excitability; exocytosis; extracellular matrix
ABSTRACT
Changes in the amplitudes of signals conveyed at synaptic contacts
between neurons underlie many brain functions and pathologies. Here we review the
possible determinants of the amplitude and plasticity of the elementary postsynaptic
signal, the miniature. In the absence of a definite understanding of the molecular
mechanism releasing transmitters, we investigated a possible alternative interpretation. Classically, both the quantal theory and the vesicle theory predict that the amount
of transmitter producing a miniature is determined presynaptically prior to release and
that rapid changes in miniature amplitude reflect essentially postsynaptic alterations.
However, recent data indicates that short-term and long-lasting changes in miniature
amplitude are in large part due to changes in the amount of transmitter in individual
released packets that show no evidence of preformation. Current representations of
transmitter release derive from basic properties of neuromuscular transmission and
endocrine secretion. Reexamination of overlooked properties of these two systems indicate that the amplitude of miniatures may depend as much, if not more, on the Ca2⫹
signals in the presynaptic terminal than on the number of postsynaptic receptors
available or on vesicle’s contents. Rapid recycling of transmitter and its possible adsorption at plasma and vesicle lumenal membrane surfaces suggest that exocytosis may
reflect membrane traffic rather than actual transmitter release. This led us to reconsider
the disregarded hypothesis introduced by Fatt and Katz (1952; J Physiol 117:109 –128)
that the excitability of the release site may account for the “quantal effect” in fast
synaptic transmission. In this case, changes in excitability of release sites would contribute to the presynaptic quantal plasticity that is often recorded. Synapse 47:
184 –199, 2003. © 2002 Wiley-Liss, Inc.
INTRODUCTION
Both the presynaptic secretion of chemical transmitters and their transduction into charge movement
across the postsynaptic membrane contribute to the
efficacy of the elementary chemical signal transmitted
from one neuron to the next. Since the earliest studies
by Fatt and Katz (1952), our understanding of transmitter release derives from studies on miniature
postsynaptic signals (potentials or currents) occurring
in the postsynaptic cell in the absence of overt action
potential activity invading the presynaptic cell and/or
axon. The vesicle hypothesis predicts that vesicles
gauge the amount of transmitter that generates a miniature signal (the quantum) and that vesicular exocytosis is the mechanism by which this quantum is entirely discharged in the synaptic cleft following Ca2⫹
entry. In less than 100 ␮s, transmission occurs between juxtaposed patches of pre- and postsynaptic
©
2002 WILEY-LISS, INC.
membranes that are about 20 nm apart and exhibit
less than 1 ␮m2 of area. Such time and space confinement may explain why the molecular basis of fast
transmission is still largely a mystery and why its
current interpretation borrows concepts from two
model systems that are more experimentally accessible
than synapses in the central nervous system (CNS).
These systems are neuromuscular transmission and
endocrine secretion. Interestingly, only the basic properties of these two systems have been incorporated into
the current representation of fast transmitter release
at central synapses. In the absence of a definitive un*Correspondence to: Jean Vautrin, INSERM U254, 71, rue de Navacelles,
34090 Montpellier, France. E-mail: [email protected]
Received 18 June 2002; Accepted 1 August 2002
DOI 10.1002/syn.10161
Published online 00 Month 2003 in Wiley InterScience (www.interscience.
wiley.com).
PRESYNAPTIC QUANTAL PLASTICITY
185
derstanding of the mechanism linking the presynaptic
Ca2⫹ signal to fast transmitter release, it is useful to
review several overlooked features of the two systems
and to examine how these properties could be relevant
to the efficacy and plasticity of elementary transmissions at synapses in the CNS. While many presynaptic
proteins have been identified, their functions still remain “largely unclear” (Augustine et al., 1999), encouraging investigation of an alternative release mechanism, which, like exocytosis, was also originally
proposed by Fatt and Katz (1952).
POSTSYNAPTIC TRANSDUCTION
A number of studies at certain CNS synapses have
suggested that all the postsynaptic receptor channels
(PRCs) are saturated by a quantum of transmitter (see
Auger and Marty, 2000, for review). In this case, the
amplitude of the postsynaptic miniature signal would
not depend on the size of the transmitter packet but on
the number of PRCs that are available on the postsynaptic membrane facing the release site (Fig. 1A). However, if postsynaptic receptors were saturated there
would be no evidence that presynaptic secretions were
quantal, in the sense that secretions would still have to
be brief to explain transient postsynaptic events, but
the amounts secreted would have little if any effect on
the amplitude of the postsynaptic events. The function
of individual presynaptic terminals would then be reduced to the role of time marker with eventual stochastic properties. However, additional studies have shown
that like neuromuscular cholinergic PRCs (Land et al.,
1980), glutamatergic PRCs (see Auger and Marty,
2000; Hanse and Gustafsson, 2001; Ishikawa et al.,
2002) and GABAergic PRCs (Vautrin et al., 1994; Perrais and Ropert, 2000; Rumpel and Behrends, 2000) at
many synapses are not all saturated during transmission. Furthermore, it has been shown that the number
of postsynaptic receptors is controlled by postsynaptic
vesicle traffic (Bayne et al., 1984; Haucke, 2000). Thus,
at many synapses some of the variations in miniature
amplitudes reflect changes in the amount of transmitter in quanta (Fig. 1B–D). In addition, receptors may
not all be available because they are regulated (Soderling and Derkach, 2000) or desensitized (Overstreet et
al., 2000) by “ambient” transmitter remaining in the
cleft (Nicholson et al., 2000) (Fig. 1D). For a given
number of postsynaptic receptor/channels activated by
a presynaptic discharge of transmitter, the postsynaptic current is defined by the elementary conductance
and kinetics of the channels, the resting membrane
potential, and the gradient of the ions permeating the
PRCs.
QUANTAL TRANSMISSION
In 1952, Fatt and Katz described miniature endplate potentials occurring in the absence of a motor
Fig. 1. Schematic representation of potential determinants of the
variability in miniature postsynaptic signals. A: In the event that all
the postsynaptic receptor channels (PRCs) are activated (open motifs,
otherwise black when closed) by the transmitter molecules (dots) from
a quantal secretion, then the miniature amplitude does not depend on
the size of the transmitter packet but on the number of PRCs available on the postsynaptic membrane facing the release site. B: In the
event that PRCs are not all activated by the quantum of transmitter,
and that exocytotic release is all-or-none, the number of transmitter
molecules released varies with the vesicle contents. C: In the event
that presynaptic exocytosis is not all-or-none, the number of transmitter molecules released may depend on the size of the fusion pore or
the duration of its opening. D: The amplitude of the miniature is also
affected by the number of PRCs desensitized (black rectangles) by the
concentration of “ambient” transmitter remaining in the synaptic
cleft. E: The amplitude of the presynaptic Ca2⫹ signal generated by
Ca2⫹ entry and release from internal store(s) may regulate the number of transmitter molecules released and the amplitude of the corresponding postsynaptic miniature (see text).
action potential together with axonal action potentialevoked end-plate potentials that “vary in a step-like
manner, corresponding to units of the miniature….”
Fatt and Katz measured miniature amplitudes and
found that “…when a large series of measurements was
tested…, the sizes were usually found to be scattered in
an approximately normal manner around a simple
mean value.” Thus, Fatt and Katz proposed that miniatures resulted from the release of “multimolecular
packets,” or quanta of the transmitter acetylcholine.
Quanta could be released either spontaneously or syn-
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J. VAUTRIN AND J.L. BARKER
of the “discharge” of one transmitter quantum generating one postsynaptic miniature (Fig. 2A). This is the
original hypothesis, which we will revisit in the context
of more recent findings. In this representation the quasi-simultaneous excitation of many release sites triggered by the invasion of the terminal by an action
potential discharges multiple quanta, thereby producing a multi-quantal postsynaptic signal. It was, however, not clear why the excitability of individual release
sites did not propagate antidromically to the rest of the
motoneuron as action potentials usually do. Indeed,
Fatt and Katz wondered: “why such ’neurogenic’ activity does not lead to a back-firing into the main motor
axon and, … to a total discharge of the motor unit.”
Furthermore, the model did not explain either the nature of the molecular coupling between the local excitation and the discharge of transmitter. At any rate, in
this representation the relatively standard size of the
release sites (or the release mechanisms they comprise)
and their all-or-none activity accounted for the standard amount of transmitter in a quantum and the
standard distribution of miniature amplitudes. Like
action potentials, presynaptic excitations may either
repeat unchanged or vary dynamically, depending on
the previous history of activity and so explain the occurrence of either similar or more variable miniatures.
VESICLE HYPOTHESIS
Fig. 2. Alternative hypotheses to explain the “quantal effect.” A:
In the “active zone hypothesis” originally proposed by Fatt and Katz
(1952), the neuromuscular junction is composed of a series of release
sites that are somehow excitable. The excitation of a single release
site (supposedly Na⫹ entry) is coupled by a yet unknown mechanism
to the release of a quantitatively related packet of transmitter molecules producing a miniature postsynaptic signal. Excitation of numerous active zones by the axonal action potential leads to the release of
as many transmitter packets. B: In the “vesicle hypothesis” later
proposed by del Castillo and Katz (1954, 1955) at the neuromuscular
junction, a miniature results from the release of the transmitter
contents from one vesicle following exocytosis. C: Vesicles are also
found in presynaptic terminals throughout the central nervous system (CNS). It is broadly accepted that exocytotic events underlie
postsynaptic miniatures in the CNS. D: Recent studies revealed that
all miniatures are not associated with the fusion of a vesicle with the
plasma membrane (Stevens and Williams, 2000) but are associated
with a presynaptic Ca2⫹ signal (Emptage et al., 2001). Thus, according to a revised version of the original hypothesis formulated by Fatt
and Katz, the quantal effect may result from a direct coupling between the presynaptic Ca2⫹ signal and transmitter release.
chronously when triggered by the presynaptic action
potential.
ACTIVE ZONE HYPOTHESIS
Miniatures and quantal transmission raised the
question of the origin of the quantal packets. Interestingly, in his original work on miniatures with Fatt
(1952), Katz considered that “some terminal spots of
the motor nerve endings are spontaneously active and
release ACh.” In Katz’s earliest model, a spontaneous
excitation limited to a single release site was the cause
Two years after Fatt and Katz (1952) proposed that
release site excitability accounts for quantal release,
del Castillo and Katz (1954) recorded miniatures under
conditions that eliminated Na⫹/K⫹ action potentials.
This led Katz to abandon the “active zone hypothesis”
and search for a new explanation for the standard size
of ACh quanta and neuromuscular miniature postsynaptic signals. Del Castillo and Katz (1955) proposed
“…to imagine a mechanism by which each particle
[vesicle] loses its charge of ACh ions in an all-or-none
manner when it collides with, or penetrates, the membrane of the nerve terminal. Such a mechanism of
release would offer an attractive explanation of the
’quantum’ effect which is responsible for the occurrence
of a single miniature….” In del Castillo and Katz’s
revised model, it was the rather standard size of presynaptic vesicles that accounted for the standard size
of transmitter quanta and miniatures (Fig. 2B). Miniatures were seen as resulting from the abrupt release
of all the transmitter molecules from a vesicle undergoing exocytosis (Fig. 2B,C). The quantal content was
then the number of vesicles simultaneously undergoing
exocytosis. However, in the vesicle hypothesis of quantal transmission, it is not yet clear what mechanism
limits the number of vesicles undergoing exocytosis to
one or a few per synaptic contact, as has often been
reported (Korn et al., 1994; Auger and Marty, 2000;
Kriebel and Keller, 2000).
PRESYNAPTIC QUANTAL PLASTICITY
VESICLE TRAFFIC
Since del Castillo and Katz’s proposal, much physiological evidence of increased endo- and exocytotic activities accompanying intense release of transmitter
provided further support for the dynamic involvement
of vesicle traffic in transmitter release (Heuser et al.,
1979; Gillespie, 1979; Betz and Bewick, 1992). Despite
controversial results (Marchbanks, 1996; Dunant and
Israel, 2000), and due to the absence of any alternative
explanation for miniatures, Katz’s attractive second
proposition invoking one vesicle per miniature gradually gained widespread popularity. Thus, it is now
widely believed that transmitter quanta are prepared
in vesicles and that each miniature is produced by an
all-or-none exocytotic event allowing a quantum of
transmitter to diffuse to the PRCs. However, “despite
all the evidence in its favour, final confirmation of the
one-vesicle hypothesis [one vesicle fusion generating
one miniature] will only be obtained with knowledge of
the transmitter contents of single packets and imaging
of vesicle during release” (Korn et al., 1994). Thorough
investigations of the molecular mechanisms that could
underlie exocytotic events underlying fast transmitter
have led some investigators to acknowledge that, for
now, there are “only reasonable hypotheses coupled
with negative data” (Südhof, 2000).
CNS MINIATURES
By analogy with spontaneous miniatures at the neuromuscular junction (NMJ), spontaneous small postsynaptic signals recorded in CNS neurons in the absence of
action potentials (generally due to the addition of tetrodotoxin, TTX) have been called miniatures and have
been considered as sufficient evidence for a quantal
form of synaptic transmission similar to that transmitted at the NMJ. However, contrary to normally distributed NMJ miniatures (Fatt and Katz, 1952) the amplitude distribution of the miniatures recorded in CNS
neurons “…is typically highly skewed, with most
events squeezing into the first amplitude bins, while a
significant proportion exhibit amplitudes that are 10 or
20 times higher than the mean.” (Auger and Marty,
2000; but see also Stevens, 1993, and Vautrin et al.,
1993). Thus, CNS miniatures clearly do not exhibit
standard amplitudes and do not form a normal, bellshaped amplitude distribution characteristic of quantal events, as originally defined by Fatt and Katz
(1952). Interestingly, long-term changes in miniature
properties such as those recorded during long-term
potentiation (Manabe et al., 1992) exhibit similar characteristic amplitude vs. rise-time relationship as do
fluctuations in these properties from a miniature to the
next. Given that the quantal theory formulated from
neuromuscular transmission predicted that transmitter quanta were standard, the larger variability in
successive CNS miniatures and the skewed amplitude distribution were, for a long time, believed to
187
reflect variations across synaptic sites on CNS neurons and to variable accuracy in recording such miniature signals.
VARIABLE QUANTA
However, recordings of the activity at a single synaptic site (for reviews see Vautrin and Kriebel, 1992;
Stevens, 1993; Auger and Marty, 2000) showed that
miniature variability was archetypical and was the
same between successive miniatures occurring from
the same site or from different sites. This made it
difficult to explain very rapid variations in the amplitude of successive miniatures in postsynaptic terms.
Curiously, while exocytotic secretion was thought to
explain the standard size of miniatures at the NMJ,
the larger variability in the amplitudes of miniatures
at CNS synapses did not lead investigators to challenge
the notion of an exocytotic mode of fast secretion. Pairs
recording of “double-sided” synapses where the same
quantum simultaneously generates miniatures in both
neurons with correlated amplitudes provided striking
evidence that CNS miniature variability reflected variations in the amount of transmitter released (Vautrin
et al., 1994; Frerking et al., 1995). Miniature variability has critical physiological consequences since the
trigger of an AP in the postsynaptic neuron and eventually the encoding of some neurosensory messages
depend on the amplitude and rise-time properties of
the miniatures (Locke et al., 1999). The high variability
in the amplitude of skew-distributed miniatures (sminiatures) recorded at CNS synapses was invoked to
explain the absence of clear peaks in some distributions of action potential-evoked postsynaptic currents
without challenging the idea that transmitter release
was quantal since it was accepted to be a consequence
of the well-accepted exocytotic nature of the release
mechanism. However, s-miniatures lack the very properties that Fatt and Katz (1952) used to interpret neuromuscular miniatures as quantal signals and later,
del Castillo and Katz (1954), as single exocytotic
events.
MULTIPLE SECRETORY EVENTS
In fact, the skewed distribution of CNS miniature
amplitudes itself was quite often multipeaked. Secondary peaks in distributions corresponding to two-,
three-, or many-fold the amplitude of the modal peak
(Edwards et al., 1990; Ropert et al., 1990; Fedulova et
al., 1999) were explained by proposing that some miniatures may have resulted from a burst of several secretions rather than from a single secretory event (SE)
(Figs. 3A,B, 4B,C). The composite nature of secretions
producing miniature signals was sometimes challenged
but it is supported by the prolongation of the rising
phase of many of the larger miniature currents. Such a
prolongation of the rising phase can only be explained
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J. VAUTRIN AND J.L. BARKER
Fig. 3. Miniature signal complexity. A: Diagrammatic representation of superimposed miniatures like those found at most synapses in
the central nervous system. Miniature signals are thought to be
generated by either a single (1) or a short burst of (2, 3, or more)
secretory events (SEs). B: Frequency distribution of miniatures like
those illustrated in A. The distribution is skewed (s-miniatures) because a majority of miniatures exhibit small amplitudes, and fewer,
larger amplitudes. This is often attributed to a predominance of
miniatures generated by a single SE (1) with the contribution at a
lesser frequency of miniatures composed of several (2, 3, or more) SEs.
Inserts are representative miniatures. Thick horizontal lines delineate rise time duration. C: Superimposed miniatures eventually generated by more SEs (1 to 5) than in A. D: Frequency distribution of
s-miniatures like those in C. The distribution shows a larger mean
amplitude (arrow) than in B due to an increased proportion of miniatures generated by multiple SEs. Dotted line superimposes the
distribution shown in B and dotted arrow indicates B mean amplitude. E: Diagrammatic representation of superimposed miniatures
like those found at adult neuromuscular junctions (NMJ). At adult
resting NMJs there is a large proportion of miniatures many (⬃7–12)
fold the amplitude of smaller, “subminiatures.” F: Frequency distribution of miniatures like those illustrated in D. The distribution
exhibits two main modes due to the generally minor contribution of
s-miniatures (small mode) similar to those in A and B and the major
contribution of miniatures roughly forming a bell-shaped distribution
(b-miniatures) (large mode). The bell-shaped distribution was shown
to result from miniatures generated by multiple and synchronous SEs
similar to those generating s-miniatures (see text).
in terms of extended duration of secretion (Van der
Kloot, 1995) or short delays between individual SEs
(Vautrin and Barker, 1995). The tendency for SEs to
clump in time has long been known (Fatt and Katz,
1952) but it is only recently that complex miniatures in
the CNS are seen as resulting eventually from several
SEs. Complex miniatures are now often considered evidence of “multivesicular release” (Fig. 4D) (Korn et al.,
1994; Frerking et al., 1997; Auger and Marty, 2000).
However, the “multivesicular” interpretation follows
from the assumption that each secretory event requires
Fig. 4. Regenerative Ca2⫹ signaling as the basis for miniature
complexity. A: Schematic representation of the signal triggering
transmitter release. The signal is a transient increase in the presynaptic cytosol Ca2⫹ concentration ([Ca2⫹]c) represented by dots. The
signal is initiated by the opening of a Ca2⫹ channel due to either a
depolarization of the presynaptic membrane (evoked release) or to a
moderate increase in baseline [Ca2⫹]c (spontaneous release). Ca2⫹
channel activation may propagate from one channel to the next either
by localized membrane depolarization or by Ca2⫹-induced-Ca2⫹-entry
and/or -release. The recruitment of an increasing number of neighboring channels (from left to right) is the basis of the presynaptic
excitability. B: Diagrammatic representation of the timing of the
secretory events (SEs) corresponding to steps illustrated in A. Each
channel (or group of interdependent channels) activated produces one
SE. C: Illustration of the postsynaptic miniatures generated by the
SEs represented in B. The more Ca2⫹ channels interact, the larger is
the postsynaptic miniature. Ca2⫹ diffusion underlies the progressive
recruitment of Ca2⫹ channels which may explain the variable level of
interdependency between SEs. D,E,F: Illustrations of possible bases
for multiple SEs. A miniature composed of three SEs may result
either from three exocytotic events (D), or three consecutive secretions from the same standing vesicle (E) or three Ca2⫹ activations of
another release mechanism (F).
a complete exocytotic event. This assumption is attractive but still remains unproven since it is unclear
whether the complex secretions were produced by several vesicles undergoing quasi-simultaneous exocytotic
fusions or by several pulses of secretion from the same
vesicle due to the flickering of the fusion pore, as seen
for endocrine exocytosis (Alvarez de Toledo and Fernandez, 1990; Breckenridge and Almers, 1987) or to
some other mechanism (Fig. 4D) (nonexocytotic release, see Fatt and Katz, 1952, and below). Therefore,
in the absence of further evidence compound CNS miniatures should simply be interpreted as resulting from
complex or multiple SEs.
NEUROMUSCULAR SUBMINIATURES
In fact, skew-distributed miniatures (s-miniatures)
are not restricted to CNS synapses, but have also been
reported at the NMJ together with normally distributed miniatures (bell-shaped distribution or b-miniatures) (for review see Kriebel, 1988) (Fig. 3D,E). Although the proportion of s-miniatures was usually only
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PRESYNAPTIC QUANTAL PLASTICITY
a few percent in healthy unstressed and mature NMJs,
it could predominate during synaptogenesis, regeneration, intense secretion, and in many conditions challenging or stressing the nerve terminal (Kidokoro,
1984; Kriebel, 1988; Vautrin and Kriebel, 1991). Therefore, since CNS miniatures were generally recorded
either in neuronal cultures with ongoing synaptogenesis or in brain slices with traumatized neurons, NMJ
and CNS miniatures may not be as different as sometimes believed (Auger and Marty, 2000). Like those
recorded in the CNS, s-miniatures recorded at the NMJ
exhibited a slower and more variable rising phase and
had a much smaller modal (most frequent) amplitude
than “standard” b-miniatures (Kriebel, 1988; Vautrin
and Kriebel, 1991, 1992). Thus, the typical amplitude
vs. rise-time relationship of both central and peripheral s-miniatures, which cannot reflect either transmitter diffusion or signal attenuation, may be easily explained by fluctuations from one miniature to the next
in the number of, and interval between, SEs (Vautrin
and Barker, 1995). In this interpretation, the same
release mechanism may generate either b- or s-miniatures depending on the number of SEs and their level
of synchronization. This is consistent with the presence
of both types of miniatures at the same NMJ and the
variation in their relative proportions under different
physiological or experimental conditions.
PEAKY MINIATURE EPP DISTRIBUTIONS
That miniatures may be produced by several SEs is
critical since it “would require a significant revision of
our concepts about the release process” (Stevens, 1993).
Even in 1952, when describing miniature endplate potentials at the NMJ for the first time, Fatt and Katz
looked for “a grouping of the individual sizes…. In some
experiments, this appeared to be the case, especially
when external records were obtained” (p 124). However, Fatt and Katz found that “…when large series of
measurements was tested …, the sizes were usually
found to be scattered in an approximately normal manner around a simple mean value.” Thus, although Fatt
and Katz found reasons to suspect subquantal fluctuations in miniature amplitude they were unable to
characterize either “humps or secondary peaks” in amplitude distributions, possibly because of insufficient
resolving power and/or long-term stability of recording
conditions. Since then, only a few groups have paid the
experimental price to collect datasets with both sufficient signal-to-noise ratio and sample size. The improvement in the signal-to-noise ratio was achieved
either by selecting miniatures occurring at a single site
(as detected using an additional extracellular electrode, see Wernig and Stirner, 1977), or by recording in
very small diameter muscle fibers (with much higher
input impedance such that the same quantal current
generated miniatures with several millivolts amplitude, see Matteson et al., 1981), or increasing the ionic
driving force by holding the membrane potential at
⫺140 mV (in voltage clamp mode) thereby increasing
the miniature current amplitude (see Erxleben and
Kriebel, 1988; see also Muller and Dunant, 1987). In
the absence of improved resolution of miniature amplitudes many causes may hide the existence of preferred
values. This may explain why some studies concluded
“that there are few, if any, data that directly support
the subunit hypothesis” for miniatures recorded at the
neuromuscular junction (Magleby and Miller, 1981).
Magleby and Miller (1981) suggested objectively testing the regular spacing of the peaks in order to find out
if they actually reflected subquantal fluctuations. Such
tests (autocorrelation and frequency analysis of histograms) have been performed (Csicsaky et al., 1985;
Vautrin, 1986) and have demonstrated that peaks in
amplitude distribution of sufficiently resolved miniatures were indeed often regularly spaced, demonstrating that they corresponded to preferred amplitudes and
not to random sampling variation (Fig. 3E).
TOO PEAKY?
Magleby and Miller (1981) considered that even if
the peaks in the histograms of miniature amplitudes
were statistically significant, even so they should not
be considered as evidence that miniatures were composed of multiple subunits. They pointed out that “to
see a repeating peak pattern throughout a histogram if
the mean miniature epp were composed of about fourteen subunits the coefficient of variation of the subunits would have to be less than about 2%.” Indeed,
Magleby and Miller’s (1981) resolution of miniature
amplitude did not exceed 2%. The argument developed
by Magleby and Miller and addressed to one of us (J.V.)
by Katz (pers. commun., 1986) was that biological processes cannot have such low variability. Therefore,
Magleby and Miller considered that it was “unlikely
that the variation in subunit amplitude would be small
enough,” and decided “that any repeating peaks which
extend throughout histograms must be due to random
variation in the data or some unexplained factors”
(1981). Thus, putative unidentified artifacts were invoked to dismiss the data that did not fit the classical
all-or-none nature of miniatures that should follow
from their vesicular definition. However, very low variability may be found at various levels in biological
systems. For example, at the molecular level the
amount of charges flowing through an ion channel in
an open state remains extremely constant (Sun et al.,
1999). At the cellular level, the amplitude of action
potentials triggered at sufficient interval shows no discernable fluctuations. At the system level, some species
of electric fish can fire action potentials at extremely
regular frequency with a coefficient of variation much
less than 2% (see Moortgat et al., 2000). Thus, there is
no objective argument to categorize as artifact the presence of preferred amplitudes in b-miniature distribu-
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tion recorded at high resolution as foreseen by Fatt and
Katz in 1952. Nevertheless, the subunit composition of
neuromuscular miniatures has not become “popular”
probably because there was no explanation for the phenomenon (Charles Stevens, pers. commun.). When
s-miniatures and subminiature fluctuations were reported at the neuromuscular junction, b-miniatures
were already believed to reflect the all-or-none release
of the entire contents of one vesicle, thus leaving no
clear morphological correlate for subminiature SEs.
MINIATURE PLASTICITY
Consequently, until 10 years ago, the hypothetical
immutability of the transmitter quantum routinely led
to the classical interpretation of long-term changes in
miniature amplitude in terms of changes in postsynaptic properties (see, for example, Manabe et al., 1992).
The more recent assessment of the variability in the
size of the quantum of transmitter led to proposed
explanations that, nevertheless, remain generally
within the framework of an exocytotic basis of quantal
secretions. Variations in the size of the vesicle (Sulzer
and Edwards, 2000) and variable levels of vesicle filling
and partial release of vesicle contents (Rahamimoff
and Fernandez, 1997; Sulzer and Pothos, 2000) (Fig.
1B–D) have been proposed to account for the variability
of the quantum of transmitter that is now known to
frequently underlie miniature variability. Interestingly, changes in the mean amplitude of s-miniatures
are generally not due to a change in all amplitudes
including the mode, but instead to a change in the
proportion of larger miniatures, those likely to be generated by several SEs. This cannot be simply explained
by altered postsynaptic efficacy but is consistent with a
change in proportion of miniatures generated by several SEs (Vautrin and Barker, 1995) (Fig. 3B,C). This
latter possibility, which has recently been addressed
(Llano et al., 2000), could not be considered when all
miniatures were boldly assigned to a single SE
(Manabe et al., 1992). Changes in miniature amplitude
are generally accompanied by changes in the duration
of the rising phase, which is expected from changes in
the degree of synchronization of underlying SEs (Vautrin and Barker, 1995). At the NMJ, dynamic and
reversible interconversions between the proportions of
b- and s-miniatures (Vautrin, 1992; Vautrin et al.,
1993; Kriebel et al., 1996) suggested that changes in
the degree of interaction among subminiature SEs supported both b- and s-miniatures. In this framework, the
variability in successive miniatures may reflect stochastic fluctuations in the interactions between SEs.
PRESYNAPTIC EXCITABILITY
Many (Korn et al., 1994; Vautrin et al., 1995; Llano
et al., 2000) have proposed that the spread of the presynaptic Ca2⫹ signal from one release site to the next
may be the link between interacting SEs (exocytotically or otherwise generated) contributing to the same
miniature (Fig. 4A). Indeed, Ca2⫹ channels (L-, N-, or
P/Q-type) directly triggering transmitter release are
concentrated at the transmitter-release face of fast
transmitting nerve terminals (Heuser et al., 1974; Robitaille et al., 1990; Haydon et al., 1994; Dunant, 2000;
Sand et al., 2001; Fisher and Bourque, 2001). These
channels interact positively like other voltage-gated
cationic channels since their activation depolarizes the
membrane locally, increasing the probability of activating similar channels in close proximity. However, two
mechanisms may restrict the retrograde propagation of
the local depolarization back into the axon (Fig. 5).
Interestingly, Fatt and Katz (1952) discussed the absence of back firing when proposing that miniatures
may follow from local action potentials. First, many
Ca2⫹ channels are inactivated by the increase in the
cytosolic Ca2⫹ concentration that they generate (Stotz
and Zamponi, 2001). Second, Ca2⫹-activated K⫹ channels, which colocalize at release sites, prevent the depolarization from spreading further (Yazejian et al.,
2000; Storm et al., 2001). These properties make Ca2⫹
entry initially a regenerative and then a self-limiting
process. Indeed, local and TTX-resistant Ca2⫹ spikes
were recorded at nerve terminals (a fact often overlooked) (Noebels and Price, 1977; Brigant and Mallart,
1982; Mallart, 1985) (Fig. 5).
INTRACELLULAR Ca2ⴙ
In addition, “The amplification of a [Ca2⫹] influx
signal by release of internal Ca2⫹ in neurons is very
similar to the process of excitation-contraction coupling
in cardiac muscle” (Berridge, 1998). Ca2⫹ sparks result
from transient release of Ca2⫹ from internal stores.
Ca2⫹ is stored in presynaptic terminals, in the secretory granules and presynaptic vesicles (Mitchell et al.,
2001; Parducz and Dunant, 1992), and Ca2⫹ release
from intracellular stores mediates synaptic plasticity
(Seymour-Laurent and Barish, 1995; Llano et al., 2000;
Emptage et al., 2001). Intracellular inositol trisphosphate receptor- and ryanodine receptor-coupled Ca2⫹
channels are present in presynaptic terminals (Berridge, 1998). L-type Ca2⫹ channels that are known to
interact directly or indirectly with ryanodine receptorchannels in cardiac, skeletal, and arterial muscle cells
were recently found at presynaptic terminals where
they appeared to initiate the chain reaction of channel
openings (Fig. 5) (Sand et al., 2001; Flink et al., 2001;
Chen and Grinnell, 2001; Urbano et al., 2001). It is also
known that there is a “functional coupling between
ryanodine receptors and L-type calcium channels in
neurons” (Chavis et al., 1996), probably due to the
overlap of their cytosolic Ca2⫹ microdomains (Narita et
al., 2000). Miniatures are accompanied by presynaptic
Ca2⫹ signals even when they are recorded in the absence of extracellular Ca2⫹ (Emptage et al., 2001), sug-
PRESYNAPTIC QUANTAL PLASTICITY
191
gesting that all elementary transmissions are driven
by Ca2⫹ transients. Thus, the original proposal by Fatt
and Katz that a miniature may reflect the excitability
of a single release site should be reconsidered in light of
these recent observations on presynaptic Ca2⫹ transients. In its revised formulation the local regenerative
signal is not an action potential due to a positive feedback of the membrane potential on Na⫹ entry, but
rather, on Ca2⫹-induced Ca2⫹ entry and Ca2⫹-induced
Ca2⫹ release mechanisms acting complementarily to
produce a transient peak in Ca2⫹ concentration (Figs.
2D, 4A, 5).
Ca2ⴙ-DRIVEN RELEASE
Fig. 5. Presynaptic axonal and terminal excitability. In the upper
right-hand side is a scheme representing the ionic channels involved
in the production of the axonal action potential (I) and the presynaptic
Ca2⫹ signal (II and III). Left side and bottom diagrams with corresponding roman numbers illustrate the mechanisms generating these
signals. (I) Upon sufficient membrane depolarization, voltage-dependent Na⫹ channels (Nav) open faster that K⫹ channels (Kv), which
increases further the depolarization. This positive feedback intensifies until all the local Nav open and the depolarization eventually
reaches and tops at the vicinity of the equilibrium potential for Na⫹.
Delayed activation of Kv later repolarizes the membrane. In the meanwhile this local action potential shifts all of the mobile cytosolic
charges, thereby depolarizing the neighboring zone of membrane
where the same process eventually develops. (II) There are typically
no voltage-dependent Na⫹ channels at the presynaptic membrane
but, rather, voltage- and calcium-dependent Ca2⫹ channels (Cav/Ca)
and calcium-dependent K⫹ channels (KCa). The local depolarization
produced by the current from the axonal action potential initiates
Ca2⫹ entry, which activates further CaVm/Ca channels. Ca2⫹ entry is
likely to eventually self limit due to the inhibition of the CaVm/Ca by
high [Ca2⫹]I. Ca2⫹-activated K⫹ channels (KCa) limit the depolarization of the terminal membrane. (III) Activation of intracellular Ca2⫹
channels (CaCa) by a moderate increase in [Ca2⫹]I leads to Ca2⫹
release from intracellular store(s) that complements (evoked release)
and eventually initiates (spontaneous release) the presynaptic Ca2⫹
signals (Emptage et al., 2001). Ca2⫹ release from intracellular store(s)
may initiate the presynaptic Ca2⫹ signals when basal [Ca2⫹]c is raised
and intracellular Ca2⫹ channels (CaCa) are “spontaneously” and asynchronously activated. The microdomains of Ca2⫹ is due to a bidirectional interaction between Ca2⫹ entry (II) and Ca2⫹ release (III). The
critical point is that the presynaptic excitability does not follow from
a positive then negative feedback of the membrane potential as occurs
in the axonal action potential, but, rather, by feedback of [Ca2⫹]c on
Ca2⫹ entry and/or release. Activation of KCa by Ca2⫹ release or entry
produces a K⫹ current, which is sometimes used to monitor the Ca2⫹
signals. In experimental and pathological situations limiting the function of KCa channels, the presynaptic terminal Ca2⫹ excitability becomes apparent by triggering antidromic action potentials (Mallart et
al., 1991) reminiscent of Fatt and Katz’s (1952) anticipation of possible antidromic activation of the whole motor unit.
There are results suggesting that the size of the
transmitter quantum depends on the amplitude of the
presynaptic Ca2⫹ signal. The presynaptic Ca2⫹ signal
evoked by the action potential at a single release site of
a cultured NMJ showed extremely small fluctuations
from trial to trial (DiGregorio et al., 1999), which is
reminiscent of the small variability of action potentialevoked b-miniatures at the NMJ (Fatt and Katz, 1952;
Kriebel et al., 1982). Thus, b-miniatures may be triggered by quantal (standard size) presynaptic Ca2⫹ signals. Indeed, elementary Ca2⫹ signals due to release
from internal stores may be either quantal or more
variable, perhaps then reflecting “activation of different size channel clusters and variable recruitment of
channels within a cluster” (Thomas et al., 1998). The
presynaptic Ca2⫹ signal evoked by the action potential
at a single presynaptic bouton in the CNS “showed
trial-to-trial variability and occasional apparent failures despite the faithful conduction of the action potential” (Frenguelli and Malinow, 1996). These authors
proposed that the fluctuations in the Ca2⫹ signal could
account for some of the known fluctuations in transmitter release at such synapses. In this regard, agents
altering the contribution of intracellular Ca2⫹ stores
affect the amplitude of the miniature (Llano et al.,
2000; Striker and Simkus, 2000). Furthermore, it was
recently shown that there is a quantitative relationship between the amplitudes of individual Ca2⫹ signals
in a single presynaptic terminal bouton and the amplitudes of the corresponding postsynaptic currents
(Kirischuk et al., 2000a,b). S-miniatures, which are
composed of relatively more variable transmitter discharges, may then reflect a more erratic propagation of
the local Ca2⫹ signal from one (or one cluster of) Ca2⫹
channel(s) to the next at the same release site. Although these results were not interpreted in these
terms, they are consistent with a direct coupling of the
Ca2⫹ signal to transmitter release with a Ca2⫹-dependent, rather than a vesicular definition of transmitter
discharges (Fig. 4A,B). Thus, in the framework of
Katz’s original “active zone hypothesis,” the “quantal
effect” may in fact result from quantal presynaptic
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J. VAUTRIN AND J.L. BARKER
Ca2⫹ signals, and more variable s-miniatures, from
more variable Ca2⫹ signals.
ENDOCRINE SECRETION
Hitherto, in the absence of an alternative to a vesicular definition of quanta, it has been assumed that
Ca2⫹ activates transmitter release for fast transmission by triggering exocytosis. Indeed, nerve terminals
exhibit Ca2⫹-dependent endo- and exocytotic activity.
However, the nature of the coupling of the Ca2⫹ signal
to transmitter release is still unclear (Augustine,
1999). Recently, it was proposed that exocytosis may
not be directly activated by Ca2⫹, but rather through
phosphorylation of protein kinases C and/or A, that are
themselves Ca2⫹-dependent (Hille et al., 1999). In addition, exocytosis is not necessarily an all-or-none phenomenon (Abillos et al., 1997). Thus, the Ca2⫹ signal
may control transmitter release by controlling the
amount of transmitter diffusing out of the vesicle lumen through the fusion pore (Haller et al., 2001) (Fig.
1C). In fact, there is no evidence that the transmitter
actually diffuses freely out of vesicles, as generally
assumed. In that matter the case of endocrine secretion
is instructive. “The release of transmitter amines is
quantal, …. However, there are experimental observations, which do not fit in with this version of an exocytotic release theory. Observed quantitative discrepancies could be explained if the release mechanism
allowed a fractional release of transmitter amine from
several vesicles instead of the total evacuation of a
few…. The matrices of amine storing granules (i.e.
from mast cells, chromaffin cells and nerve terminals)
show the properties of weak cation exchanger materials,…” (Uvnas, 1991). Indeed, “…vesicles may have
mechanisms to restrict the release of their contents
even though membrane fusion has occurred…. Thus,
the relationship between fusion of vesicle-plasma
membrane and the actual release of the secretions is
far from obvious” (DeFelice, 1996). In fact, “…vesiclecell fusion, revealed by cell capacitance measurements,
is temporally dissociated from secretion measured amperometrically” (Travis and Wightman, 1998). In pituitary endocrine cells, when exocytosis was activated
using high K⫹ rather than the natural secretagogue
agonist dopamine, “the cores [of the granules] were
exposed to the extracellular fluid for several minutes,
but were never released” (Angleson et al., 1998) (Fig.
6A). Paramecium defense also involves Ca2⫹-activated
exocytosis with Ca2⫹ entry and Ca2⫹ release from intracellular submembranous stores (Erxleben et al.,
1997). In this case of so-called “frustrated exocytosis,”
there is a “clear separation of exocytotic membrane
fusion from any later Ca2⫹-dependent steps of the secretory cycle” (Klauke et al., 1998).
Fig. 6. Alternative mechanisms for endocrine secretion and transmitter release. A: In endocrine cells, not all secretory products (dots)
are in solution. Rather, a significant proportion is bound to charges on
a core matrix (mesh). Stimulation of both exocytosis and matrix decondensation (1) are required to allow all the secretory products to
diffuse in the extracellular medium. Stimulation of exocytosis alone
(2) may not lead to significant release but eventually to re-endocytosis
of the core matrix and the matrix-bound secretory products (see
Angleson et al., 1999). B: Likewise, two models may explain the rapid
recycling of extracellular transmitter at the synapse without always
evidence of vesicle membrane fusion with the plasmalemma. Between
two successive rounds of calcium-activated release, “kissing” vesicles
(1) may remain connected by the fusion machinery to the plasma
membrane and may simply refill rather than fuse and recycle. Rapid
refill of the vesicles requires transmitter molecules to be rapidly
transported back in the cytoplasm first, then, in turn, transported into
docked “kissing” vesicle (see text). Alternatively, vesicles may first
undergo complete exocytosis (2) and fuse with the presynaptic plasma
membrane prior to transmission (therefore after previous transmission). The transmitter may then remain bound to the surface matrix
now located on the extracellular side of the presynaptic membrane.
Some of these molecules may be set free by the action of the intracellular Ca2⫹ signal on an integral membrane molecular assembly to be
identified. After transmission, transmitter molecules are rapidly recaptured and immediately incorporated in the readily releasable pool
at the presynaptic surface.
FRUSTRATED PRESYNAPTIC EXOCYTOSIS?
Frustrated exocytosis may be seen as a reversed
endocytic uptake after adsorption on the membrane
surface (see Morimoto et al., 1995). Thus, immediate
release may not follow solely from exocytosis. Stevens
(1999) said about frustrated exocytosis in pituitary
cells (Angleson et al., 1999): “If something like this
regulation by post-fusion vesicular properties also is
found in the small vesicles responsible for fast synaptic
PRESYNAPTIC QUANTAL PLASTICITY
transmission, the classical concept of quantal release
will have to be revised.” Indeed, the physiological role
of the presynaptic terminal does not involve long-distance diffusion of sufficient number of signaling molecules, as is the case for endocrine secretions. Rather,
presynaptic terminals transmit an immediate signal
across a space that is less than 20-fold the length of
transmitter molecules and that is full of charged molecules (Ledeen, 1993; Bignami et al., 1993; Wheatley,
1998). “The clear appearance of synaptic vesicles in the
electron microscope seems to be misleading” (Rahamimoff and Fernandez, 1997). The idea that transmitter
molecules are stored as freely diffusing molecules inside presynaptic vesicles (as proposed by Whittaker,
1982) may be incorrect since free diffusion is likely to
be very restricted in highly structured and charged
environments such as the vesicle and the synaptic cleft
(Bignami et al., 1993; Whittaker and Kelic, 1995;
Wheatley, 1998; Benson et al., 2000). For example,
Ca2⫹ ions, which are quite diffusible in water, do not
diffuse freely in the cytoplasm (Llinas et al., 1992). It is
thus not clear whether transmitter molecules are free
to diffuse within vesicles and, later after exocytosis, in
the synaptic cleft. In this regard, presynaptic vesicles,
in addition to transmitter, are endowed with a matrix
of highly negatively charged proteoglycan-bound glycosaminoglycans, sialoglycoproteins, and sialoglycolipids (gangliosides) lining the lumenal side of their membrane. Most of these charges are sialic acid radicals.
Ledeen et al. (1993) found that “Synaptic vesicle membranes had the highest overall concentration [of sialic
acid] relative to protein, but a concentration approximately comparable to that of presynaptic membranes
when expressed relative to phospholipid.” In fact, sialoglycoconjugates have been proposed to act as chromogranins in endosecreting granules, assisting in the
sequestration of ACh in Torpedo electric organ presynaptic vesicles (Whittaker and Kelic, 1995). It is an
undeniable physical fact that ionic charges bound on a
support, such as those in ion-exchanging resins, impose
an electrostatic gradient that affects the distribution of
otherwise freely diffusing ions (Oshshima and Ohki,
1985; Gunther et al., 1997). The phenomenon is similar
and comparable in amplitude to that imposed by ionic
charges not permeating through the cell membrane
and responsible for the resting potential.
PRESYNAPTIC GLYCOCALYX
Contrary to secretory granules where chromogranins
are released, the negatively charged molecules (sialoglycolipids and sialoglycoproteins) lining the lumens of
presynaptic vesicles at fast transmitting synapses are
not released because most are integral components of
the vesicle membrane and, after vesicle fusion, become
the surface of the plasma membrane itself. Thus, sialic
acid, hyaluronic acid, and heparan sulfate radicals
eventually contribute to the presynaptic extracellular
193
surface matrix (Kuhn et al., 1988; Whittaker and Kelic,
1995; Carlson, 1996). “The ectodomains of glycolipids
and of integral membrane proteins, along with any
bound or adsorbed molecules, constitute the cell surface coat called the glycocalyx” (Schnitzer, 1988). In the
brain, “the extracellular space appears empty by electron microscopy because hyaluronic acid is readily dissolved during the preparation…” (Bignami et al.,
1993). However, “Electron microscopy of cationic colloidal iron-stained ultrathin sections revealed that the
synaptic boutons were separated from each other by
the proteoglycan matrix” (Ohtsuka et al., 2000). Thus,
due to the direct contact of pre- and postsynaptic glycocalyx, there is “solid state” continuity between preand postsynaptic membranes (see Philips et al., 2001).
It is also known that membrane or membrane-bound
adhesion molecules, eventually brought to the cell surface by exocytosis, are involved in synaptic plasticity
(Zanetta, 1998; Saghatelyan et al., 2000; Rafuse et al.,
2000; Son et al., 2000). In addition, it is a general
phenomenon that “…glycosaminoglycans mediate the
interactions with a variety of extracellular ligands …”
(Tumova et al., 2000).
JUXTACELLULAR TRANSMITTER
Few studies have addressed the possibility of interaction of transmitter molecules with the presynaptic
glycocalyx. However, neuropeptides were found to interact with glycolipid receptors (Valdes-Gonzalez et al.,
2001). Another study “…suggested that enzymatic destruction of a part of the glycocalix of cells forming the
neuromuscular junction and of a part of the extracellular matrix results in a weakening of the nonspecific
acetylcholine binding” (Vinogradova and Matiushkin,
1998). Furthermore, Diamond and Jahr (1997) found
that glutamate was buffered in the synaptic cleft and
exogenous ganglioside treatment decreased glutamate
exocytoxicity for PC12 cells (Cunha et al., 1997). We
found that GABA molecules adsorb at the surface of
liposomes containing the trisialoganglioside GT1b
(Vautrin et al., 2000; see also DeFeudis et al., 1980),
which is localized in synaptic vesicles and plasma
membranes at synapses throughout the CNS. Extracellular GABA also colocalized with GT1b ganglioside
at the surface of neurons during the differentiation of
their processes (Vautrin et al., 2000). Interestingly, the
presence of a juxtacellular accumulation of GABA was
also detected by recording the autocrine activation of
GABAA receptor/Cl- channels on neurons differentiating
in culture (Valeyev et al., 1993; 1998). After the formation
of functional synapses, postsynaptic GABAergic transient currents (reflecting “quantal release”) together
with tonic postsynaptic currents (due to ambient transmitter in the cleft) could be depressed or eliminated by
renewing or stirring, respectively, the extracellular saline bathing the neuronal culture (Melnick et al., 1999;
Vautrin et al., 2000). These surprising effects on tonic
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J. VAUTRIN AND J.L. BARKER
and transient GABAergic signals were immediate
(Vautrin et al., 2000), suggesting that the readily releasable pool (RRP) of transmitter was juxtacellular
(Fig. 6B). Data suggested that ambient transmitter in
the cleft was in equilibrium with surface juxtacellular
transmitter and that quanta were immediately formed
from this surface-accessible pool of transmitter.
REVISITING PRESYNAPTIC
VESICLE TRAFFIC
The role of vesicle traffic in reloading rapidly a surface-accessible, juxtacellular pool of transmitter is consistent with the persistence of transmission, although
with a reduced RRP, in the absence of vesicle traffic
(Henkel and Betz, 1995; Becherer et al., 2001). The role
of the vesicle traffic in the regulation of the RRP is also
supported by many experiments in which the RRP was
regulated by protein kinase C-dependent vesicle traffic
(Ghirardi et al., 1992; Stevens and Sullivan, 1998;
Becherer et al., 2001). While in an increasing number
of experiments presynaptic vesicle traffic and transmitter release did not correlate well (Henkel and Betz,
1995; Stevens and Williams, 2000; see also Dunant,
2000, for review) another, plausible role for presynaptic
vesicle traffic in transmitter release is emerging. Exoand endocytotic vesicle traffic at presynaptic terminals
(like elsewhere) may work as a conveyor belt recycling
specific components of the membrane and membranebound molecules (e.g., adhesion and neurotrophic molecules, transporters etc.) at specific sites on the cell
surface (Boudier et al., 1999; Bagnat et al., 2000; Prinetti et al., 2000; Nichols et al., 2001; Chavis and
Westbrook, 2001). “Cholesterol-sphingolipid microdomains (lipid rafts) are part of the machinery ensuring
correct intracellular trafficking of proteins and lipids.
The most apparent roles of rafts are in sorting and
vesicle formation, although their roles in vesicle movement and cytoskeletal connections as well as in vesicle
docking and fusion are coming into focus. … Important
clues have also been uncovered about the mechanisms
coupling raft-dependent signaling and endocytic uptake” (Ikonen, 2001; see also Schubert, 1989; and Herreros, 2001 for neurons).
TRANSPORT INTO VESICLES
“Classical transmitters are stored into secretory vesicles by an active transporter driven by a vacuolar-type
H⫹-ATPase” (V-ATPase) (Gasnier, 2000). Blocking either the transporter (Reimer et al., 1998; Travis et al.,
2000) or the “energization” of the membrane by the
H⫹-ATPase (Zhou et al., 2000; Hong, 2001) depresses
synaptic transmission. In addition, transporter expression affects quantal size (Reimer et al., 1998; Sulzer
and Pothos; 2000). All this is consistent with quantal
transmitter release requiring initial loading of vesicles
prior to engaging in an exocytotic form of transmitter
release. However, “awareness of plasma membrane energization by V-ATPases provides new perspectives”
(Wieczorek et al., 1999). “A growing body of recent
evidence supports the possibility that the stimulusdependent recycling of transporter-carrying vesicles
can alter the abundance of transporters in the plasma
membrane…” (Park et al., 2000). The ACh transporter
located in the vesicle membrane “becomes part of the
nerve-terminal membrane as the vesicle fuses into the
plasma membrane” (Smith, 1992). Indeed, “biochemical studies identified two binding sites for vesamicol [a
specific blocker of the vesicular ACh transporter]; one
is the vesicular acetylcholine uptake system and the
other is the vesamicol binding protein localised in the
nerve terminal membrane. This vesamicol binding
component seems to play an important role in the frequency-dependent modulation of the evoked release of
transmitter quanta” (Maeno et al., 1994). Most importantly, previously accumulated transmitter levels in
synaptic vesicles are not maintained in the absence of
active transport (Carlson and Ueda, 1990; Zhou et al.,
2000; see also Uvnas, 1991). This suggests that vesicles
may not work as tight closed containers preloaded with
defined amounts of transmitter, but rather that transmembrane voltage and pH gradients serve to retain
transmitter molecules, possibly by ion exchange, at the
vesicle lumenal surface and this tethering of transmitter may persist at the presynaptic membrane surface
after vesicle fusion (Fig. 1E, 6B).
COINCIDENCE OF EXOCYTOSIS
WITH TRANSMISSION
The vesicular origin of quanta and the exocytotic
procedure of their release has been based on coincidences between exocytotic events and synaptic transmissions. However, the best timing of substructural
alterations within the pre- and postsynaptic membranes during synaptic transmission suggested that
exocytosis at motor nerve terminals (Dunant, 2000)
like in endocrine cells (Travis and Wightman, 1998;
Barg et al., 2002) may follow rather than precede
transmitter release, which is consistent with exocytosis
being involved in restoring the presynaptic terminal
properties after challenging transmitter release.
Matching the number of vesicles and the number of
miniature transmissions has been used to demonstrate
the vesicular basis of quanta. However, we have seen
that there is now evidence that individual miniature
signals may result from several SEs. Most importantly,
there are now reports that miniature postsynaptic signals may occur in the absence of evidence of vesicle
fusions (see below and Henkel and Betz, 1995; Stevens
and Williams, 2000).
RECYCLING
After fusion with the plasma membrane, components
of vesicle membranes are recycled by endocytic process
PRESYNAPTIC QUANTAL PLASTICITY
(Heuser and Reese, 1973; Prior and Clague, 1997).
“Following endocytosis, synaptic vesicles are then shuttled back into the vesicle pool, where they briefly mix
with other vesicles, become immobilized, and remain
gelled with their neighbours, even while moving en
masse again to the presynaptic membrane as a prelude
for another round of exocytosis” (Betz and Angleson,
1998). Contrastingly, extracellular transmitters, transmitter analogs or transmitter precursors gain rapid
access to the RRP such that the most recently taken up
or synthesized is preferentially released (Large and
Rang, 1978; Solis and Nicholl, 1992; Dunant and Israel, 2000). “Transporters buffer synaptically released
glutamate on a submillisecond time scale” (Diamond
and Jahr, 1997). “Sustained high frequency synaptic
transmission requires re-uptake of glutamate from the
synaptic cleft. … With repeated challenges, the ability
to release neurotransmitter recovered 10-fold more
rapidly than restoration of [vesicle fusion characterized
by] FM2-10 de-staining” (Bains and Staley, 2000). The
rapid recycling of transmitter in the absence of evidence of exocytosis is generally explained by a putative
“rapid reuse of readily releasable pool vesicles …” (Pyle
et al., 2000). To explain the discrepancy between the
vesicle traffic and the transmitter traffic it is being
proposed that some vesicles remain docked at the release site and refill rapidly on site between each putative Ca2⫹-controlled discharge of transmitter (Stevens
and Williams, 2000; Südhof, 2000; Valtorta et al., 2001)
(Fig. 1C).
IS TRANSMITTER RECYCLED
OR SHUTTLED?
The idea that some vesicles may release quanta
through a “kissing” or “standing” mode rather than
undergoing a complete exocytotic fusion follows from
the notion that vesicles, as structural entities, are absolutely required for quantal transmission since they
are believed to determine the amount of transmitter in
quanta and that some form of exocytosis is the mechanism that releases transmitter quanta in the synaptic
cleft. Indeed, in endocrine systems it has been shown
that secretory granules may “kiss-and-run.” In this
case only a small fraction of the granule’s content is
released, namely, the fraction that is not adsorbed in
the matrix and free to diffuse. Contrastingly, miniatures recorded with and without evidence of vesicle
fusion are indistinguishable (Stevens and Williams,
2000), suggesting that synaptic vesicles may not “kiss”
like endosecretory granules. We have reviewed arguments supporting the original Fatt and Katz (1952)
hypothesis that quanta may not correspond to vesicle
contents as was originally hypothesized but rather may
reflect the amplitude of the presynaptic Ca2⫹ signal as
has been reported (Kirischuk et al., 1999a,b). Moreover, there is experimental evidence that quanta are
not preformed (Elmqvist and Quastel, 1965; Large and
195
Rang, 1978; Chang et al., 1998; Vautrin et al., 2000).
The issue is thus about how short the reloading cycle of
the RRP is. We found in neuronal culture that the
depression of quantal GABA release by a stream of
saline was preceded by a decrease in the tonic GABAergic signal. The clearing of ambient transmitter off the
cleft was confirmed by a resensitization of postsynaptic
GABA receptors prior to the blockade of transient signals. These observations suggested that ambient transmitter in the cleft was required for the formation of
quanta. In addition, immediately (5 sec) following introduction of a synthetic GABA analogue (isoguvacine)
into the synaptic cleft, all the postsynaptic transients
recorded for minutes thereafter were mediated by the
analog without any evidence of further GABAergic
quanta (Vautrin et al., 2000). These results suggested
that quanta were formed from a RRP of surface-accessible transmitter rather than from an intracellular pool
(cytoplasmic or vesicular) (Fig. 6B). Logically, this
leads to an extension of the “kissing vesicle” hypothesis. Since the internal surface of vesicles may retain
transmitter molecules like the core of secretory granules (Whittaker and Kelic, 1995), and since this property may persist after vesicle fusion with the presynaptic plasmalemma (Vautrin et al., 2000), then
reloading of the release machinery following release
may not result from a recycling via the cytoplasm involving transport across plasma and vesicle membranes but, more simply, via a direct and immediate
reloading with transmitter directly from the cleft. In
this case, successive transmissions are mediated by a
shuttling of transmitter between the presynaptic membrane surface and the receptor/channels on the
postsynaptic membrane.
REVERSE TRANSPORT
The mechanism by which transmitter is released
either directly from a (fusing or kissing/standing) vesicle or from a juxtacellular compartment has not been
elucidated. Many hypotheses were formulated to explain how exocytosis could account for fast synaptic
transmission within the vesicle hypothesis (Südhof,
2000). Although transporters for fast-acting neurotransmitters like glutamate and GABA have been well
characterized for their uptake properties, there is also
strong evidence from a variety of preparations that
transporters may function in “reverse” mode to release
transmitter (Levi and Raiteri, 1993; Richerson and
Gaspary, 1997; Beckman and Quick, 2000).
In fact, transporters are found to “act to maintain a
constant level of neurotransmitter at the synaptic cleft”
(Bernstein and Quick, 1999; see also Katagiri et al.,
2001). Thus, reverse transport in addition to exocytosis
and recapture may supply the surface-accessible pool of
transmitter. We recently found that a well-established
blocker of the GABA transporter GAT1, SKF89976a
(Vautrin and Barker, unpubl.) rapidly blocked both
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J. VAUTRIN AND J.L. BARKER
continuous and quantal release of GABA in embryonic
hippocampal neurons differentiating in culture in the
absence of astrocytes. In these conditions, ambient
transmitter in the cleft was free to exchange with the
diffusion sink represented by the bath saline. It is thus
likely that in such conditions GABA was continuously
transported toward the cleft to maintain the RRP,
thereby explaining why blockade of the transport immediately blocked both tonic and fast transient transmissions.
RELEASE
Whether release is exocytotic or from the surface, the
molecular mechanism coupling the Ca2⫹ signal to the
liberation of transmitter molecules remains unclear.
Transporters were often judged too slow to account for
fast transmission. However, if the presynaptic Ca2⫹
signal triggers the conditions that either reverse many
transporter proteins synchronously, or transiently interrupt the binding of GABA to the presynaptic glycocalyx sites, then the synchronous release of transmitter
molecules from the presynaptic surface may be the
basis of fast transmitter release and transient postsynaptic signals. Thus emerges a new model capable of
explaining both a Ca2⫹-controlled complex transient
secretion and a very rapid recycling of a small RRP of
quanta sometimes occurring in the absence of evidence
of vesicle traffic (Fig. 6B). However, neither in a vesicular, nor in a Ca2⫹ signal definition of the quantum is
the release exocytotic per se in the sense that it does
not correspond to the all-or-none fusion of a vesicle
with the presynaptic plasma membrane as in Katz’s
second hypothesis.
In the well-established interpretation derived from
Katz’s vesicle hypothesis, the transmitter is released
through a Ca2⫹-controlled transient opening of a pore
between the lumen of a docked vesicle and the extracellular space. It assumes that the secretion is due to
the diffusion of transmitter in solution from within a
vesicle and that the vesicle may be very rapidly refilled
between two successive rounds of opening of the pore.
For unknown reasons, the vesicle would be refilled
preferentially with cytosolic transmitter recently either synthesized or taken up.
Alternatively, and in the framework of Fatt and
Katz’s earliest proposition that release sites are excitable, the Ca2⫹ signal accounts for the kinetics of the
secretions of transmitter from a juxtacellular pool
ready for release. The transmitter partitions between a
surface-adsorbed phase and an ambient phase of transmitter diffusing generally at low concentration in the
cleft. Fast transmission then follows from an intracellular Ca2⫹-activated transient decrease in the affinity
of the transmitter for some presynaptic surface binding
sites, increasing momentarily the concentration of ambient transmitter molecules. Then the role of exocytosis
is to rapidly recycle juxtacellular RRP with vesicular
storage pool together with the release machinery.
CONCLUSION
When considering the full extent of the models used
to decipher fast synaptic transmission in the CNS, it
appears that 1) miniatures are not unitary but complex; 2) quantal release is a more dynamic process than
suggested by the presynaptic ultrastructure; and 3)
Ca2⫹ dynamics, at least as much as exocytosis, control
SEs. It will be necessary to take into consideration all
the physiological properties of quantal transmission to
identify the molecular link coupling the presynaptic
Ca2⫹ signal to fast transmitter release and to understand the basis of its plasticity and its pathologies.
NOTE ADDED IN PROOF
Since this paper was accepted Zenisek et al. published an article in Neuron (2002 35:1085–1097) showing that at the retinal bipolar cell terminal FM1-43
leaves synaptic vesicles in milliseconds. This further
supports the idea that miniatures occurring in the absence of loss of vesicular FM1-43 (Südhoff, 2000) might
not result directly from exocytotic release. Also, a paper
by Reigada et al. “Control of neurotransmitter release
by an internal gel-matrix in synaptic vesicles”, shows
that this matrix regulates the availability of free diffusible acetylcholine.
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