11618 Journal of Physiology (2001), 532.3, pp.

637–647 637

Repetitive nerve stimulation decreases the acetylcholine

content of quanta at the frog neuromuscular junction

Ligia A. Naves and William Van der Kloot

Departments of Physiology and Biophysics and of Pharmacological Sciences, State
University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA
(Received 24 August 2000; accepted after revision 22 December 2000)
1. We investigated how elevated quantal release produced by motor nerve stimulation affects the
size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which
excitation–contraction coupling was disrupted. Two hundred stimuli reduced the size of the
time integrals of the miniature endplate currents (∫MEPCs), measured at the same junction
immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When
the solution contained 10 µM neostigmine (NEO) 3000 stimuli reduced ∫MEPCs by 60 %, because
with acetylcholinesterase (AChE) inhibited, ∫MEPC size is more sensitive to changes in
acetylcholine (ACh) content. Similar decreases in miniature endplate potential size (∫MEPP)
followed repetitive stimulation of contracting preparations.
2. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000
nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less
ACh released per quantum.
3. Following 3000 stimuli at 10 Hz the sizes of the ∫MEPCs increased back to pre-stimulus values
with a half-time of 8–10 min. Recovery was blocked by (_)-vesamicol (VES), by
hemicholinium-3 (HC3) and by nicotinic cholinergic agonists – all of which inhibit ACh loading
into synaptic vesicles.
4. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide
m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from
unstimulated controls. After resting for hours following stimulation, the releasable number
increased, even when ACh loading inhibitors were present.
5. We conclude that the inhibitors do not block a significant fraction of the ACh loading into
reformed reserve vesicles and propose that ACh can be loaded in a series of steps.
Most chemical synapses release their neurotransmitter in stimulation reduces quantal size did not establish whether
packets, or quanta. Quantal release produces a high local the size was smaller because less ACh was released or
concentration of the transmitter in the synaptic cleft, because the endplate was less sensitive to ACh (Doherty et
which enables the synapse to operate rapidly (reviewed al. 1984; Glavinovi´c, 1988). We set out to clarify these points.
by Van der Kloot & Molgó, 1994). The quanta are
packaged within synaptic vesicles. After the quanta are The present paper follows on recent work in which the rate
released the membrane of the synaptic vesicles is of quantal release was accelerated with elevated K+ (Van
retrieved and formed into new vesicles, which are then der Kloot et al. 2000). Because elevated K+ solutions depress
reloaded with transmitter. These recycled vesicles then choline (Ch) uptake into nerve terminals, they cannot be
become available for release. Within this broad picture, used to see whether release itself decreases quantal size
many details of the retrieval and reloading processes (Naves et al. 1996). Two methods used in the elevated K+
remain fuzzy (Betz & Angleson, 1998). work are used again: inhibiting ACh loading into synaptic
vesicles and measuring the store of releasable quanta.
A large body of previous work concluded that repetitive Three inhibitors of ACh loading were used. (_)-Vesamicol
stimulation does not alter the size of the quanta at the (VES) blocks active ACh transport into isolated synaptic
neuromuscular junction (reviewed by Van der Kloot & vesicles (reviewed by Prior et al. 1992; Parsons et al. 1993;
Molgó, 1994). Contrary reports that repetitive nerve Van der Kloot & Molgó, 1994). NH4+ inhibits ACh uptake
638 L. A. Naves and W. Van der Kloot J. Physiol. 532.3

by diminishing the proton gradient across the vesicular Use of neostigmine

membrane that is required for transmitter accumulation
(Van der Kloot, 1987). Hemicholinium-3 (HC3) blocks the
Na+-driven Ch uptake mechanism in the membrane of
the motor nerve terminal, which may be directly coupled
to ACh synthesis and loading (Yamamura & Snyder,
1973; Gylys & Jenden, 1996).
The number of vesicles in the releasable store was
estimated by counting how many quanta were released
by the proton ionophore carbonyl cyanide m-chloro-
phenylhydrazone (CCCP) (Molgó & Pécot-Dechavassine,
1988). The CCCP experiments showed that the number of
quanta released from different preparations varied
substantially. However, the numbers released by CCCP
from paired preparations from the same frog are
correlated, which means that the muscle from one side of
the body can be used as a control for the other (Van der
Kloot et al. 2000). Therefore CCCP was used in the
present paper to see whether there are fewer quanta in
the releasable store after prolonged repetitive
stimulation, as reported by Ceccarelli & Hurlbut (1980).
The results are discussed in light of the recent discovery
that there are two distinct endocytic pathways for
reforming synaptic vesicles at the frog neuromuscular
junction (Richards et al. 2000).
METHODS
Preparations
Sartorius or cutaneous pectoris nerve–muscle preparations were
dissected from Rana pipiens, which were killed by double pithing in
accordance with University guidelines (Van der Kloot et al. 1998).
The Ringer solution contained (mM): NaCl, 120; KCl, 2.0; CaCl2, 2.5;
Tes–NaOH, 4.0 (pH 7.4). Many of the recordings were made in
Ringer solution containing 0.03 µM tetrodotoxin, to suppress
spontaneous muscle contractions.
NEO (10 µM) was used in many experiments because it was found
that inhibiting much of the AChE accentuates experimentally
induced changes in ∫MEPC sizes (Van der Kloot et al. 1994). This is
predicted by mathematical modelling of the generation of MEPCs in
the presence and absence of AChE. We used the model of Wathey et
al. (1979), which ignores the anatomical complexities of the endplate
included in more sophisticated models (Stiles et al. 1996), which for
present purposes should make little difference. The equations, the
methods used for solving them and the parameters used are in Van
der Kloot et al. (1994). Eliminating AChE has little effect on the peak
amplitudes of the model MEPCs (Fig. 1A), but has a substantial effect
on their time integrals (Fig. 1B). Without AChE the plot of MEPC
size as a function of quantal ACh content has a steeper slope.
Therefore with AChE inhibited, the changes in the ∫MEPCs reflect
changes in quantal ACh content better than changes in amplitude
(see also Van der Kloot, 1997).
Electrophysiology
When indicated in the text, nerve–muscle preparations were pre-
treated by soaking in Ringer solution containing 2 M formamide for
17–20 min to largely eliminate excitation–contraction coupling (del
Castillo & Escalona de Motta, 1978). The preparations were pinned on
a layer of silicone rubber in an acrylic chamber with a volume of
about 5 ml. Recordings were at room temperature (18–22 °C).
The motor nerve was drawn into a suction electrode for stimulation.
The pulses were at least twice supra-maximal, 80 µs square waves,
which in most experiments were produced by an FHC pulsar 6B
stimulator (Brunswick, ME, USA) driving a WPI A360 stimulus
isolator (Sarasota, FL, USA).
MEPCs were recorded using the two-electrode voltage clamp; the
recording methods and criteria for accepting data were described in
detail by Van der Kloot et al. (1994). Briefly, the glass micro-
electrodes were bevelled, with DC resistances between 2 and 4.5 MΩ.
Clamping was done with an Axon Instruments Axoclamp-2A voltage
clamp amplifier (Foster City, CA, USA). The signals were passed on
to an Axon Instruments CyberAmp 300 signal conditioner, which
amplified over a bandwidth from 0.1 to 1000 Hz. The signal
emerging from the CyberAmp was split, one branch going to an Axon
Instruments AI 2020 event detector, the other to a ComputerBoards

Figure 1. Calculations of model MEPCs with and without active AChE

1, with the enzyme active; 0, with the enzyme inhibited. A, the peak amplitudes; B, the time integrals.
With the integrals the slope of the relation between the number of molecules in the quantum and the
magnitude of the response is steeper when there is no enzyme.
J. Physiol. 532.3 Stimulation decreases quantal size at the neuromuscular junction
DAS-16 330 A/D converter (Mansfield, MA, USA). When a MEPC
passed a threshold set just above the noise level, the event detector
delivered a TTL pulse to the A/D board, which digitized at 10 kHz.
The 200 points (2 ms) before the TTL pulse and the 800 (8 ms) after
the pulse were output to a computer. An operator observed the
signal. If the MEPC was not contaminated by an overlapping MEPC
or by electrical noise, its voltage–time integral, ∫MEPC, was
calculated as the measure of the quantal size (Van der Kloot, 1997). If
the holding current began to markedly increase during an
experiment the clamp was discontinued and the data discarded.
MEPPs and ∫MEPPs were recorded with the same apparatus, except
that only a single intracellular electrode was used. The amplitudes of
the MEPPs were corrected to a standard resting potential of _90 mV
by the method of Katz & Thesleff (1957). Examples of the MEPPs
and MEPCs were shown previously (Van der Kloot, 1991; Naves &
Van der Kloot, 1996).
Estimating the releasable store of quanta with CCCP
Briefly, the number of quanta released following exposure to CCCP
was measured as follows (details in Van der Kloot et al. 2000). An
endplate was penetrated. CCCP was added to the bath to give a final
concentration of 10 µM. Every 100 s the computer recorded a series of
A/D points at 2 kHz extending for 0.64 s. If the membrane potential
of the fibre fell to a level where seeing the MEPPs became
problematical the microelectrode was inserted into a fresh endplate.
In some sets all recording was from a single fibre, in others as many
as three junctions were penetrated. Previous work showed that the
number of quanta released from paired muscles was correlated,
regardless of how many penetrations were required (Van der Kloot et
al. 2000). Recording was terminated when the miniature endplate
potential frequency (FMEPP) reached a low value, usually < 1 s_1.
After the recording was completed, the operator reviewed the
records in the form of 64 ms segments, counting the number of
MEPPs in each segment. The total number of MEPPs released was
estimated assuming that the measured rate persisted until the next
set of measurements was taken in.
Measuring evoked quantal output
To test for possible effects of a drug on evoked quantal output
nerve–muscle preparations were placed in a solution containing
(mM): NaCl, 120; KCl, 2.5; MgCl2, 2.5; CaCl2, 0.2; and TES, 4.0. This
solution substantially decreased quantal output. Mean quantal
outputs (m0) were estimated by the method of failures, counting the
number of stimuli at 0.5 Hz that were not followed by an endplate
potential (n0) and the total number of stimuli (N). Then:
m0 = ln(N/n0).
The standard error of the estimate (S.E.) is given by:
1 _ (n0 /N)
S.E. = ÷—————
n0
(Martin, 1966). Differences between mean quantal outputs were
tested by Student’s t test.
Detecting subpopulations of quantal size
The methods described in this section were used and tested for
reproducibility previously (Van der Kloot et al. 2000). To perform the
test adequately, at least 300 ∫MEPPs must be recorded in each set of
data. The ∫MEPPs from each set were sorted in ascending order and
plotted as a cumulative curve. Each of these curves was fitted with
one and with two cumulative lognormal distributions, as illustrated
in Fig. 8, using the Levenberg-Marquardt method (Press et al. 1989).
If the fit to two distributions appeared to be better, the next step was
to determine whether improvement was because the two
639
distributions actually fitted the data better or merely because more
fitting parameters were used (Horn, 1987; Van der Kloot et al. 2000).
The ∫MEPPs, arranged in ascending order, were sorted into bins, each
of which contained a minimum of five observations. The predicted
values from the one lognormal and two lognormal curves were sorted
into the same bins. The likelihood functions, ll = Fobs w ln(fobs/fpred)
were calculated for each bin. These likelihood functions were then
summed, ∑llone and ∑lltwo. Then:
G = 2(∑llone _ ∑lltwo).
2
G is distributed as x with 3 degrees of freedom, because the curve
that is the sum of two lognormal distributions is calculated with three
more parameters than the single one. The probability that the data
are best described by one lognormal curve rather than two is obtained
from the x2 and the degrees of freedom
Statistics
All of the means reported in the text and in the figures are given with
the ±95 % confidence intervals, which were determined by
resampling (Van der Kloot, 1996). In the experiments on the
recovery of quantal size after stimulation, resampling was also used
to determine if there was a statistically significant difference
between the first 30–50 ∫MEPCs after the end of the repetitive
stimulation and the last 30–50 ∫MEPCs recorded at the junction (Van
der Kloot, 1996). If P < 0.05 the differences are termed significant.
Chemicals
The drugs were from Sigma (St Louis, MO, USA), except for
(_)-vesamicol (2-(4-phenylpiperidino)cyclohexanol), HC3 and
nicotine bitartrate, which were obtained from RBI (Natick, MA, USA).
RESULTS
Quantal size decreases with stimulation
Preparations pretreated with formamide to disrupt
excitation–contraction coupling were placed in Ringer
solution without NEO. Junctions were voltage clamped
and ∫MEPCs were measured. Then the clamp was
discontinued, the motor nerve was stimulated 200 or 3000
times at 10 Hz, the junction was re-clamped and 50–100
∫MEPCs were measured. Since FMEPP was elevated
following the stimulation, all of these ∫MEPCs were
recorded in less than 5 min; the short recording time is
important because, as will be shown shortly, size recovers
as time passes. There was a significant decrease in ∫MEPC
size following stimulation (Fig. 2A). The decrease in
∫MEPC size following 200 stimuli was almost identical to
the decrease in MEPP amplitude measured by Doherty et
al. (1984), who withdrew the recording electrode during
stimulation. When we repeated our measurements in the
presence of 10 µM NEO the decreases in size following
3000 stimuli were greater (Fig. 2A). This is accounted for
by a mathematical model of MEPC generation, which
shows that the slope of the plot relating the time integral
of the number of channels opened as a function of the
ACh released is steeper when AChE is inhibited (Fig. 1).
No significant decreases in ∫MEPC sizes were detected
following 50 (n = 4) or 100 stimuli (n = 4) in 10 µM NEO.
Doherty et al. (1984) showed that size decreased because
there were fewer MEPPs with large amplitudes, and an
increase in the proportion with smaller amplitudes. They
640 L. A. Naves and W. Van der Kloot J. Physiol. 532.3

Figure 2. Tetanic stimulation at 10 Hz reduces miniature sizes

A, the reduction in ∫MEPC sizes after stimulation from preparations uncoupled with formamide pre-
treatment; in each of the experiments the ∫MEPCs were recorded at the same junction before and after
the stimulation. The reductions following 3000 stimuli were greater in 10 µM NEO. 4, in Ringer solution;
$, in 10 µM NEO. In parentheses above each bar is the number of experiments. The error bars show the
95 % confidence intervals. B, the decrease in ∫MEPP size following stimulation at 10 Hz in preparations
with contraction intact; recording was in Ringer solution containing 10 µM NEO. ∫MEPPs were recorded
at randomly selected junctions before and after the stimulation. The numbers of junctions recorded from
was as follows: 0 stimuli, 11; 100 stimuli, 28; 150 stimuli, 5; 200 stimuli, 12; 300 stimuli, 6; 1500 stimuli,
16; 3000 stimuli, 5.

argued that this shows that the quanta are smaller
because less ACh is released in each packet. We will take
up this question shortly.
Because of the natural variation in quantal size at
junctions and between junctions, for accurate
measurement of size changes it is necessary to measure
from the same junction before and after treatment, as in
the experiments just reported, or to measure from five or
more fibres in each preparation before and after
treatment (reviewed by Van der Kloot, 1991). Measuring
from a number of fibres after stimulation was not feasible
in the present work, because quantal size recovers over
time (see below). The best we could do to see whether
quantal size also decreased following stimulation when
excitation–contraction coupling was intact was to
stimulate at 10 Hz and then rapidly measure ∫MEPPs
(Fig. 2B). Size surely decreased following stimulation, so

Figure 3. A period of repetitive stimulation does not reduce the endplate response to an
iontophoretic pulse of ACh
A, the larger signal, peaking at 60 ms, is an example of the depolarization produced by the ACh pulse. The
smaller signal, at 20 ms, is a MEPP. The solution contained 10 µM NEO. B, the time integrals of the
responses to ACh pulses over time. During the interruption in the recording the motor nerve was
stimulated 3000 times at 10 Hz.
J. Physiol. 532.3 Stimulation decreases quantal size at the neuromuscular junction 641

the decrease is not an artifact somehow produced by the
uncoupling in formamide.
Stimulation does not substantially decrease
sensitivity to ACh
Quantal sizes might be smaller after stimulation because
less ACh is released per quantum, because the endplate is
less sensitive to ACh, or both. Repetitive stimulation
decreases quantal size at the Drosophila neuromuscular
junction by desensitizing receptors (Adelsberger et al.
1997) and there is evidence for desensitization with
repetitive stimulation at the frog neuromuscular junction
as well (Giniatullin et al. 1989). One experimental test
used to help decide between these alternatives was to
apply iontophoretic pulses of ACh to endplates in Ringer
solution containing 10 µM NEO, the solution in which
stimulation produced the largest decreases in quantal size.
The preparations were pretreated with formamide to
uncouple contraction. The depolarization of the endplate
in response to each ACh pulse was measured as the time
integral of the endplate depolarization. After control
responses had been collected, the iontophoretic pulses
were stopped while the motor nerve was stimulated 3000
times at 10 Hz. Then the iontophoretic pulses were
resumed and the responses measured once again.
Stimulation decreased the response to ACh only slightly
(Fig. 3), even though ∫MEPCs were reduced by roughly
60 % (Fig. 2A). Similar results were obtained in five
additional experiments. This suggests that quantal size
decreases mainly because less ACh is released per
quantum. Additional evidence supporting this conclusion
will be presented shortly.
Recovery of ∫MEPC size following stimulation
Formamide-treated preparations were placed in Ringer
solution containing 10 µM NEO. ∫MEPCs were measured.
The clamp was discontinued and the motor nerve
stimulated 3000 times at 10 Hz. As soon as the
stimulation was terminated, the clamp was reestablished
and ∫MEPCs measured once again. Immediately after
stimulation the ∫MEPC size was substantially decreased
compared to before stimulation; the mean decrease was to

Figure 4. ∫MEPC size falls following tetanic stimulation and then recovers toward initial size,
unless inhibitors of ACh loading into synaptic vesicles are present
All solutions contained 10 µM NEO. The filled bars show the mean ∫MEPC size before stimulation. The
break in the x-axis indicates that the motor nerve was stimulated 3000 times at 10 Hz. The subsequent
plot shows the mean ∫MEPC size during the succeeding bins. Each bin contained 50 MEPCs, so their time
durations varied as shown. A, in Ringer solution without any inhibitor; B, in 2 µM VES; C, in 1 µM HC3;
D, in 1 µM DMPP.
642 L. A. Naves and W. Van der Kloot
50 ± 12 % (n = 24). Following stimulation quantal size
rose back toward the pre-stimulation levels over a period
of minutes (Fig. 4A). Routinely there is a considerable
spread in quantal sizes, so the means of relatively small
samples of ∫MEPCs have substantial variance. Because of
this scatter the half-time for recovery can only be
approximated; 8–10 min seems a reasonable estimate
from the 24 experiments. Doherty et al. (1984), who
recorded MEPPs in contracting preparations by
withdrawing the recording electrode during stimulation
and then reinserting in the same fibre, observed similar
time courses of recovery.
Effects of ACh loading inhibitors on the recovery of
∫MEPC size
The inhibitors were added to the solution just before the
beginning of recording, because in 10 µM NEO in
unstimulated preparations they gradually decrease
∫MEPC size by as much as 30 % (Van der Kloot et al. 1994;
Naves & Van der Kloot, 1996).
(_)-Vesamicol. The results so far suggest that quantal
size recovers because additional ACh is added to the
quanta. To test this hypothesis further, we measured
∫MEPCs in the presence of VES. Quantal size did not
recover significantly when the solution contained 2 µM
VES, but did recover in 0.2 µM (Figs 4B and 5). The
inhibition of recovery by VES supports the explanation
that quantal size decreases after stimulation mainly
Figure 5. The effects of drugs on the recovery of
∫MEPC size following 3000 stimuli at 10 Hz
Each column shows the ratio of the size in the final
time bin following recovery to the size in the initial
time bin, immediately after the stimulation. The
error bars on the right of the columns show the +95 %
confidence limits. The vertical dashed line shows the
ratio expected if there was no recovery in quantal
size. The numbers to the right of each bar show
(number of experiments in which the sizes in the final
time bins were significantly larger at
P < 0.05)*/(total number of experiments).
J. Physiol. 532.3
because less ACh is released per quantum, and that
recovery occurs when additional ACh is packed into the
vesicles.
Hemicholinium-3. HC3 at 1 µM blocked the recovery of
quantal size following the stimulation, while 0.1 µM HC3
was ineffectual (Figs 4C and 5). (In some of the experiments
with 1 µM HC3 the quantal size decreased significantly
during the recovery period following the stimulation.) Much
of the previous work on the effects of HC3 on quantal size
was done with concentrations of 10 µM or even 30 µM. Such
high concentrations should be avoided because they might
have additional effects on the preparation.
If the uptake of Ch is important for the recovery of
quantal size following stimulation, we were concerned
that the extent of the recovery might be diminished by
the anticholinesterase in the extracellular solution, which
by blocking ACh hydrolysis might materially diminish
the quantity of Ch available for recycling. Therefore we
repeated the control experiments in solutions containing
10 µM Ch. The presence of Ch did not significantly alter
the extent of the recovery (Fig. 5).
HC3 might act on another target. In snail neurons, HC3 is
an agonist for a neuronal-type ACh receptor (Poulain et
al. 1987). We can see whether HC3 is also an agonist for
the neuronal ACh receptor on the frog motor nerve
terminal because if so it will decrease evoked quantal
output (Van der Kloot, 1993). The mean quantal output,
m0, was estimated by the method of failures. In control
preparations 2 µM carbachol, an ACh agonist, decreased
Figure 6. ∫MEPP sizes grow smaller following
repetitive stimulation at 10 Hz in the presence of
inhibitors of ACh loading and 10 µM NEO
The error bars show the ±95 % confidence limits. ª, in
30 mm NH4+; 1, in 1 µM HC3; 3, in 2 µM VES;
0, with all three inhibitors together. As pointed out
in the text the significant differences between the
mean ∫MEPPs in different inhibitors after
stimulation are likely to reflect differences in the
preparations themselves, rather than treatments
given. The number of endplates recorded from ranged
from 6 to 21 for each point.
J. Physiol. 532.3 Stimulation decreases quantal size at the neuromuscular junction 643

evoked quantal output, m0, to 56 ± 11 % (n = 10; 95 %
confidence interval) of the pre-drug control. In 1 µM HC3
evoked quantal output was 128 ± 22 % (n = 10) of the
control. Clearly HC3 does not act like a nicotinic agonist
by decreasing evoked output. HC3 probably halts the
recovery of ∫MEPC size by blocking the Ch transporter.
ACh agonists. Nicotinic ACh agonists block the addition
of ACh to quanta in preparations that have been treated
to produce an increase in quantal size (Van der Kloot,
1993). In the next experiments 1 µM carbachol was added to
the extracellular solution before the repetitive stimulation.
In five experiments quantal size did not recover
significantly after the stimulation; in two of the five
experiments quantal size decreased significantly during the
recovery period (Fig. 5). Nicotine and 1,1-dimethyl-4-
phenyl-piperazinium (DMPP, an agonist which targets
nicotinic ACh receptors) also blocked the recovery of
quantal size (Figs 4D and 5).
∫MEPPs after stimulation in the presence of ACh-
loading inhibitors
Preparations were exposed to 1 µM HC3, 2 µM VES,
20 mM NH4+, or all three simultaneously. ∫MEPPs were
measured at several junctions, and then the motor nerves
were stimulated at 10 Hz for varying times. After the
stimulation ended, ∫MEPPS were measured once again. In
all the inhibitor-containing solutions, the size of the
∫MEPPs decreased with stimulation (Fig. 6). The fewest
stimuli used was 3000; more stimuli did not appear to
decrease quantal size further. The declines were very
similar in the presence of the individual inhibitors or
when all three inhibitors together were present. Even
after the longest stimulation periods, MEPPs were
readily seen and their frequency was in the normal range.
The number of quanta released by CCCP following
repetitive stimulation
To see whether the store of releasable quanta falls
following repetitive stimulation and whether quanta
containing ACh can be formed when ACh-loading
inhibitors are present, we used CCCP to estimate the store
of releasable quanta (Molgó & Pécot-Dechavassine, 1988;
Van der Kloot et al. 2000). To answer the first question
the nerve from one preparation of the pair was
stimulated 3000 times at 10 Hz. Then it was placed in
10 µM CCCP and the number of quanta subsequently
released measured. The second preparation was exposed
to 10 µM CCCP without any stimulation (Fig. 7A).
Stimulated preparations released fewer quanta than the
unstimulated controls. To see whether rest increases the
releasable store once again, one preparation was
stimulated 3000 times at 10 Hz and then kept in Ringer
solution for 4–5 h before exposure to 10 µM CCCP. The
other preparation was exposed to CCCP immediately
after stimulation (Fig. 7B). The rested preparations
released more quanta than those treated with CCCP
immediately after stimulation.
We used paired preparations once again to see whether
ACh-loading inhibitors would decrease the number of
quanta added to the releasable store during a recovery
period following repetitive stimulation. One of the pair
was stimulated 3000 times at 10 Hz in Ringer solution,

Figure 7. Changes in the number of quanta released by 10 µm CCCP as a result of stimulation,

after recovery from simulation, and after recovery in the presence of ACh-loading inhibitors
A, one muscle was unstimulated (x-axis), while the second was stimulated 3000 times at 10 Hz (y-axis)
before exposure to the CCCP. The continuous line shows the expectation if the number of releasable
quanta was unchanged. The dotted line is the regression coefficient for the data, R 2 = 0.47. B, one muscle
was exposed to the CCCP immediately after being stimulated 3000 times (x-axis), while the other was
allowed to rest for 3–5 h before exposure to CCCP (y-axis). The continuous line shows the expectation if
rest had no effect. The dotted line is the regression, R 2 = 0.70. C, preparations stimulated 3000 times at
10 Hz either without drugs or in the presence of ACh-loading inhibitors. The number of quanta released
by CCCP after 3–5 h rest was measured. The continuous line is the expectation if the drugs had no effect
on releasable number. The dotted lines are the ±95 % confidence limits for the regression. The slope of the
regression, which is not shown, was 0.82, R 2 = 0.82. 0, 2 µM VES; 1, 2µM VES + 1 µM HC3.
644 L. A. Naves and W. Van der Kloot J. Physiol. 532.3

and then allowed to rest for 3–5 h before being exposed to
10 µM CCCP and the number of quanta released
estimated. The second preparation was treated the same,
except that during stimulation and recovery the solution
contained either 2 µM VES, or 1 µM HC3 + 2 µM VES. The
inhibitors did not decrease the size of the releasable store
(Fig. 7C). Since releasable quanta are formed during the
rest period, it seems that they can be made even in the
presence of ACh-loading inhibitors.
Subpopulations of quantal sizes following stimulation
in VES
The approach in these experiments was used previously
when we enhanced the rate of quantal release with
elevated K+ in the presence of an ACh-uptake inhibitor
and then examined the ∫MEPPs to see whether there were
two size classes (Van der Kloot et al. 2000). Since there
were no obvious differences in the effects of the inhibitors
tested, all the experiments looking for two sub-
populations of quantal sizes were done in 2 µM VES. (We
stimulated at 30 Hz because we hoped that we could use
others’ data to estimate how many quanta were released
by the stimulation, but this required too many
assumptions to be useful.) Van der Kloot et al. (2000)
showed that at junctions most ∫MEPP distributions are
fitted by a single lognormal probability distribution
function. However, after 1000 or more stimuli, the
cumulative distributions of the ∫MEPPs from most of the
junctions were fitted best by two lognormal curves in 46
out of 61 cases (Fig. 8). The figure shows the data plotted
on probability scales; the classical approach for detecting
two subpopulations was by examining such plots for
breaks between two lines fitted to the plot. The figures
show how difficult this approach is in practice. In earlier
experiments in elevated K+ the fraction of the ∫MEPPs in
the larger subpopulation decreased as more quanta were
released (Van der Kloot et al. 2000, Fig. 5B). A decrease
in the fraction in the larger subpopulation was by no
means as sure in nerve stimulation experiments, perhaps
because the rate at which the number of quanta released
falls off with nerve stimulation varies from one
preparation to the next (data not shown). A decrease in
the fraction in the large subpopulation suggests that in
ACh-loading inhibitors the newly formed quanta are
smaller. The ratio of the mean size of the ∫MEPPs in the
larger subpopulation to that in the smaller was 2.6 ± 0.26
(n = 46), close to the ratio found in the elevated K+
experiments. The advantage of the K+ data is that we can
estimate the total number of quanta released by the
treatment: 60 % of the ∫MEPPs were in the larger
subpopulation when 100 000 quanta had been released.
DISCUSSION
Katz (1969), who introduced the term ‘quantum’, later
pointed out that the name misleads some readers. By a
misconceived analogy with the physical quantum, they
acquire the false idea that miniatures have almost
uniform size. In reality quantal size at a junction varies
over a fivefold range; in untreated preparations the CV is
about 0.3. With treatment, the range is much greater.
A variety of treatments substantially increase quantal
size (references in Van der Kloot et al. 1998; reviewed by
Sulzer & Pothos, 2000). In the most dramatic instances at
the neuromuscular junction, mean size increases fourfold.
At both the frog and the mouse neuromuscular junctions
size increases because more ACh is loaded into the readily
releasable pool of quanta (Yu & Van der Kloot, 1991; Van
der Kloot, 1993).
Surely it is less surprising that quanta can also be made
smaller. What is unexpected is that relatively modest

Figure 8. Examples of the fitting of the distribution of ∫MEPPs to one and to two lognormal
functions
The points are shown as black circles. Red curve, one lognormal; blue curve, two lognormals. The ∫MEPCs
were recorded after stimulation at 30 Hz in solution containing 2 µM VES. The x-axis is a probability scale;
plotting data that fitted to a normal probability distribution function on the scale produces a straight line.
J. Physiol. 532.3 Stimulation decreases quantal size at the neuromuscular junction
stimulation, releasing fewer than 20 % of the quanta in
the terminal, significantly decreases size. The best data
come from experiments in which ∫MEPC size was
measured in the same fibre before and after the
stimulation, which bypasses problems with the variations
in size from junction to junction and from preparation to
preparation or the uncertainty of reinserting electrodes
into the same fibre before and after a bout of contraction
(Fig. 2A). Size is significantly decreased by 200 stimuli.
Three thousand stimuli reduced the ∫MEPCs recorded in
10 µM NEO by roughly 60 %. Experiments in which
MEPPs from randomly chosen endplates are sampled
before and after tetanic stimulation are less reliable for
measuring the magnitude of the size changes, but they
clearly show that quantal size also decreases following
stimulation of preparations in which excitation–
contraction coupling was intact (Fig. 2B).
There are two reasons to think that after stimulation the
quanta are smaller largely because they contain less ACh.
First, the response of the endplate to ACh applied by
iontophoresis was little changed after repetitive nerve
stimulation that would decrease ∫MEPC size by 60 %
(Fig. 3). Second, after stimulation was over quantal size
gradually rose back toward the pre-stimulus value as the
preparation rested. Recovery was blocked by the
inhibitors of ACh loading into synaptic vesicles: VES,
HC3, NH4+, and nicotinic agonists (Fig. 5).
When do vesicles receive their stockpile of ACh? The
answer seems to be that loading can occur in several steps.
Two hundred stimuli at 10 Hz releases somewhere
between 10 000 and 20 000 quanta (Doherty et al. 1984;
Van der Kloot, 1993). Doherty et al. (1984) pointed out
that this is about the number of vesicles in the readily
releasable pool, probably those attached to the active
zones at the motor nerve terminals. So it seems that once
the vesicles join the readily releasable pool they receive an
appreciable supplementary loading of ACh. We called
this second-stage loading (Van der Kloot, 1991; Naves et
al. 1996; Naves & Van der Kloot, 1996).
This name should be replaced. It was based in the idea
that the readily releasable pool is replenished by vesicles
coming from the reserve pool in the axoplasm (Betz &
Angleson, 1998). Loading ACh into vesicles in the reserve
pool was the first stage and further loading in the readily
releasable pool was the second. This picture has been
redrawn by Richards et al. (2000), who show that when
stimulation first begins the vesicles in the readily
releasable pool are replenished via a discrete, fast
endocytic pathway. It requires more prolonged
stimulation to move vesicles from the reserve pool into
the readily releasable pool. A second, slower endocytic
pathway that involves membrane infoldings and
cisternae replenishes the reserve pool. Since there is no
single first stage, the term second-stage loading is
misleading. ‘Final loading’ seems a better name for the
pumping of additional ACh into the vesicles once they are
645
added to the readily releasable pool. Previous work
showed that the enlargement of quantal size occurs
largely by increased final loading, so the amount of ACh
incorporated in final loading can be regulated.
There is substantial evidence for final loading (Doherty et
al. 1984; Van der Kloot, 1991; Naves et al. 1996; Naves &
Van der Kloot, 1996). Final loading can be blocked by all
the inhibitors of vesicular ACh uptake that we have used.
The half-time for final loading is 8–10 min (Doherty et al.
1984; Van der Kloot, 1993; Fig. 4). Vesicles reaching the
readily releasable pool either via the rapid endocytic
pathway or from the reserve pool seem to receive final
loading. Quantal size is transitorily decreased following
200 stimuli, in which most of the vesicles released from
the readily releasable pool presumably are replenished by
the rapid endocytic pathway. It is also transitorily
decreased by 3000 stimuli, when many of the
replacement vesicles must come from the reserve pool,
judging from the substantial decrease in the number of
releasable quanta in the terminals (Fig. 7).
Our data give no more information about the initial
loading of ACh into the vesicles formed by the fast
endocytic pathway. They do suggest that the filling of
the vesicles in the reserve pool involves more than one
step. After prolonged stimulation in the presence of VES
two subpopulations of quantal size were often detected
(Fig. 8). Note that final loading is not operating in these
experiments because it has been blocked by the VES.
Apparently during the stimulation vesicles released from
the reserve pool are replaced by recycled vesicles
containing less ACh. This was more clearly demonstrated
in earlier work when the release rate was increased with
30 mM K+ solution. The fraction in the subpopulation of
larger size quanta decreased as more quanta were released
(Van der Kloot et al. 2000).
This leaves us with the question of how ACh first gets into
reformed, recycling vesicles in the reserve pool. Even
after thousands of stimuli in the presence of ACh uptake
inhibitors, quanta were still readily seen and measured
(Fig. 6). One possibility was that these quanta were all
loaded before the inhibitor was present, and the recycled
vesicles contain no transmitter. At the snake
neuromuscular junction empty vesicles are formed and
fuse with the terminal membrane when the preparation is
stimulated (Parsons et al. 1999). We do not know whether
this occurs in the frog. Previous work shows that
repetitive stimulation reduces the number of releasable
quanta and the number of vesicles in the terminal
(Ceccarelli & Hurlbut, 1980). After 9000 stimuli frog
nerve terminals are maximally stained with FM2-10 after
resting for 20 min (Richards et al. 2000). We estimated
the number of releasable ACh quanta by counting the
MEPPs released by CCCP treatment. The CCCP-
releasable store decreased when the drug was added
immediately following 3000 stimuli (Fig. 7A). On the
other hand, the number of releasable quanta increased
646 L. A. Naves and W. Van der Kloot
again following a period of rest, and this increase also
occurred when uptake inhibitors were present (Fig. 7B
and C). Apparently some ACh is loaded in recycled
vesicles even when an ACh-loading inhibitor is present in
a concentration adequate to block the recovery of quantal
size in final loading (Fig. 5). The initial loading of ACh
into recycled vesicles does not seem to require the VES-
inhibited ACh transporter or the proton gradient
diminished by NH4+.
The most difficult aspect of these results to account for is
the formation of reserve vesicles containing some ACh in
the presence of HC3, which might be expected to block all
ACh production in the terminal by halting Ch recycling.
We speculate that there is a pathway for ACh synthesis
that obtains Ch from another source, and that HC3 acts
because Ch reuptake, ACh synthesis and transport into
vesicles are linked into a chain (Gylys & Jenden, 1996).
More work is needed on how ACh is loaded when
inhibitors are present.
Another unresolved question is the pathway by which
nicotinic agonists depress ACh loading into recycling
quanta or into quanta that are increasing in size (Van der
Kloot, 1993), because we are accustomed to think of these
as directly operating membrane ions channels. Neurons
from the rat dorsal septal nucleus hyperpolarize in
response to nicotinic agonists (Sorenson & Gallagher,
1996). The hyperpolarization is eliminated when GTPyS
is present in the patch pipette, strongly suggesting a
G-protein link between this neuronal nicotinic receptor
and the K+ channel that is activated. Perhaps a second
messenger is also involved in the frog motor nerve
terminal as a link between the nicotinic neuronal receptor
on the motor nerve terminal and the mechanism for
loading synaptic vesicles with ACh. Motor nerve
terminals have nicotinic receptors (Tsuneki et al. 1995),
which are implicated in the control of evoked quantal
output (Van der Kloot, 1993; Tian et al. 1997). The
possibility that feedback mechanisms alter quantal
loading as well as quantal release deserves attention at
other synapses where autoinhibition occurs (reviewed by
Wu & Saggau, 1997; MacDermott et al. 1999).
The combination of final loading and a fast endocytic
pathway explains results that seriously challenged the
concept that quantal release occurs from vesicles
prepackaged with transmitter. Biochemical data show
that newly synthesized ACh is released more rapidly than
expected if it mixed with all the ACh within the terminal.
Newly synthesized false transmitters also soon appear in
the quanta (reviewed by Van der Kloot & Molgó, 1994;
Naves et al. 1996). Since most of the terminal’s total stock of
ACh is in the reserve vesicles, newly synthesized ACh or
false transmitter should be put into quanta during final
loading. Vesicles formed by the fast endocytic pathway
should be filled mostly with newly synthesized ACh.
Therefore the prompt release of newly synthesized
transmitter seems to be well accounted for.
J. Physiol. 532.3
When the link between quanta and vesicles was first
proposed, it seemed reasonable to think that vesicles were
formed, filled and then stand by until released. Now we
know there are two endocytic pathways for recycling and
several steps in transmitter loading. At least the final
step in transmitter loading can be regulated to vary the
ACh content of the quanta. The picture now lacks the
elegance of simplicity, but does account for the data.
Naturally enough, as we have pointed out, there are still
open questions to be answered.
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Acknowledgements
This work was supported by Grant 10320 from the National Institute
of Neurological Diseases and Stroke. We thank Judy Samarel for
assistance.
Corresponding author
W. Van der Kloot: The Boat House, 1 Fort Gate, Newhaven, East
Sussex, BR9 9DR, UK.
Email: wvanderkloot@post.harvard.edu
Author’s present address
L. A. Naves: Department of Physiology and Biophysics, Federal
University of Minas Gerais, Belo Horizonte, MG, Brazil.
Email: lnaves@mono.icb.ufmg.br