1T. H. Morgan School of Biological Science, University of Kentucky, Lexington 40506-0225; and 2Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084
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ABSTRACT |
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He, Ping,
R. Chase Southard,
Dong Chen,
S.
W. Whiteheart, and
R. L. Cooper.
Role of -SNAP in Promoting Efficient Neurotransmission at the
Crayfish Neuromuscular Junction.
J. Neurophysiol. 82: 3406-3416, 1999.
In this manuscript, we
address the role of the soluble N-ethylmaleimide sensitive factor
attachment protein (
-SNAP) in synaptic transmission at the
neuromuscular junction of the crayfish opener muscle. Immunochemcial
methods confirm the presence of
-SNAP in these preparations and show
that it is concentrated in the synaptic areas. Microinjection and
electrophysiological studies show that
-SNAP causes an increase in
neurotransmitter release yet does not significantly affect the
kinetics. More specific quantal analysis, using focal, macropatch,
synaptic current recordings, shows that
-SNAP increases transmitter
release by increasing the probability of exocytosis but not the number
of potential release sites. These data demonstrate that the role of
-SNAP is to increase the efficiency of neurotransmission by
increasing the probability that a stimulus will result in
neurotransmitter release. What this suggests is that
-SNAP is
critical for the formation and maintenance of a "ready release"
pool of synaptic vesicles.
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INTRODUCTION |
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The studies of neurotransmitter (NT) release have
yielded a long list of molecules, proposed to play a role in
neurotransmission and have suggested an outline of a molecular
interactions required for synaptic vesicle (SV) exocytosis (reviewed in
Bajjalieh and Scheller 1995; Martin 1997
;
Rothman 1994
; Sudhof 1995
). Membrane proteins from the synaptic vesicle [called vesicle soluble
N-ethylmaleimide sensitive factor attachment protein
receptors (v-SNAREs), e.g., vesicle associated membrane protein
(VAMP)/synaptobrevin] and from the active zone [called target
membrane SNAREs (t-SNAREs), e.g., syntaxins and synaptosome associated
protein 23 or 25 (SNAP23/25)] appear to be essential for membrane
fusion events (Hunt et al. 1994
; Littleton et al.
1998
; Weber et al. 1998
). Current data suggest
that the three SNARE proteins, interlocked by the interactions of their
coiled-coil domains (Poirier et al. 1998
; Sutton
et al. 1998
; Weber et al. 1998
), form a
bimembrane-spanning complex that is sufficient for bilayer fusion
(Weber et al. 1998
).
The interactions of the SNARE proteins are, in part, regulated by
cytosolic proteins called SNAPs and NSF
(N-ethylmaleimide sensitive factor). SNAPs initially
bind to the SNARE complex and serve as adapters to correctly position
NSF (Clary et al. 1990; Weidman et al.
1989
). SNAP is also responsible for the stimulation of NSF's
ATPase activity, which in turn is needed to disassemble the SNARE
complex (Barnard et al. 1997
; Nagiec et al.
1995
). Although SNAREs are needed for fusion (Hunt et
al. 1994
; Littleton et al. 1998
; Weber et
al. 1998
), it is less obvious what role SNAP and NSF play in
neurotransmission (Banerjee et al. 1996
; Haas and Wickner 1996
; Hay and Martin 1992
; Martin
et al. 1995
; Mayer and Wickner 1997
;
Mayer et al. 1996
). Injection studies using squid axons
have shown that SNAPs increase the output of a terminal (DeBello
et al. 1995
). Similar studies have shown that NSF increases the
efficiency of NT release (Schweizer et al. 1998
).
Studies with the comatose mutant in Drosophila
(temperature-sensitive NSF) have demonstrated that active NSF is
required for SV consumption and have suggested that NSF functions both
before and after membrane fusion (Kawasaki et al. 1998
;
Littleton et al. 1998
; Siddiqi and Benzer
1976
; Tolar and Pallanck 1998
). The model
emerging from this research suggests that NSF and SNAPs are involved in
both SNARE priming before fusion and in SNARE recycling after membrane fusion.
In this study, we used the opener neuromuscular junction (NMJ) from the
crayfish to assess the role of -SNAP on NT release. In
microinjection studies,
-SNAP increased synaptic output with only a
small effect on release kinetics. Quantal analysis indicates that
-SNAP had no effect on the total number of release sites but
significantly increased the probability that a stimulus will result in
NT release. Consistently,
-SNAP injection reduced the failure rate
for evoked NT release. These data indicate that
-SNAP is not
involved in the membrane fusion event but, like NSF (Schweizer et al. 1998
), functions to increase the pool of "ready
release" SVs at the synapse. Preliminary reports of this study have
appeared in abstract form (He et al. 1997
;
Southard et al. 1998
).
This work is in partial fulfillment of the Master of Science degree requirement for P. He.
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METHODS |
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Western blotting analysis of crayfish tissues
Crayfish tissue, consisting of the ventral nerve cord and the
identified abdominal extensor muscle (containing motor nerve terminals)
were dissected and then homogenized using a Dounce homogenizer in lysis
buffer [(in mM) 25 Tris/HCl (pH 8.0), 500 KCl, 250 sucrose, 2 EGTA,
and 0.5 1,10-phenanthroline] with 1% (vol/vol) Triton X-100. After
incubation on ice for 45 min., the insoluble material was removed by
centrifugation. The protein concentrations of the detergent-solubilized
proteins were measured using the bicinchoninic acid assay (Pierce,
Rockford, IL) with bovine serum albumin (Sigma, St. Louis, MO) as
standard. For Western blotting analysis, 150 µg of crayfish extract
was fractionated by 10% SDS-PAGE and transferred to nitrocellulose.
Blots then were probed with anti--SNAP antibodies (Whiteheart
et al. 1992
), and the immunodecorated proteins were detected by
Enhanced Chemiluminescence (ECL, Pierce) using anti-rabbit IgG
secondary antibodies conjugated to horseradish peroxidase (Sigma).
Similarly prepared, rat brain extracts were used as positive blotting controls.
Immunofluorescence
Whole-mount preparations were pinned to a silicone elastomer
(Sylgard) dish with the muscle in a stretched position. They were fixed
with 2.5% (vol/vol) glutaraldehyde, 0.5% (vol/vol) formaldehyde
dissolved in a buffer A (0.1 M sodium cacodylate, pH 7.4, 0.022% wt
CaCl2, 4% wt sucrose) for 1 h with two
changes of solution. The preparation then was placed into vials and
washed in buffer A containing 0.2% (vol/vol) TritonX-100 and 1%
(vol/vol) normal goat serum (Gibco/BRL, Grand Island, NY) for 1 h
with three changes at room temperature. The tissue then was incubated
with primary antibody to -SNAP [1:1000 in PBS buffer (in mM): 136 NaCl, 2.7 KCl, 10 Na2HPO4,
and 1.5 mM KH2PO4] in a
shaker at 4°C for 12 h. The tissue was washed three times and
incubated in secondary antibody (goat, anti-rabbit IgG conjugated with
Texas Red, Sigma) diluted 1:200 with PBS buffer at room temperature for
2 h, followed by two washes in buffer. The synaptic locations were
observed by immunocytochemistry as previously shown in nerve terminals (Cooper 1998
; Cooper et al.
1996a
). Fluorescent images of the nerve terminals (Fig.
1, A and B)
were obtained with a Nikon Optiphot-2 upright fluorescent
microscope using a ×40 (0.55 NA) Nikon water immersion
objective (Nikon, Melville, NY) with appropriate illumination.
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Preparation of -SNAP and
-SNAP(L294A) mutant
The -SNAP(L294A) mutant (Barnard et al. 1997
)
expression construct was prepared by PCR, and the point mutation was
confirmed by dideoxy sequencing. Recombinant
-SNAP and the (L294A)
mutant were expressed in Escherichia coli using the pQE-9
expression vector (Qiagen, Valencia, CA) and purified by
Ni2+ nitrilotriacetic acid agarose
affinity chromatography as described in Whiteheart et al.
(1993)
. Both SNAP proteins were dialyzed exhaustively against
injection buffer [containing (in mM) 10 HEPES/NaOH (pH 7.4), 115 KAc,
2 glutathione, 3.5 MgAc2 and 0.5 ATP and 1% (vol/vol)
glycerol] and stored at
80°C at a concentration of 0.51 mg/ml.
Crayfish NMJ preparation
All experiments were performed using the first walking leg of
crayfish, Procambarus clarkii (4-6 cm in body length,
Atchafalaya Biological Supply, Raceland, LA). The opener muscle of the
first walking legs was prepared by the standard dissection
(Cooper and Ruffner 1998; Dudel and Kuffler
1961
). The tissue was pinned out in a Sylgard dish for viewing
with an Optiphot-2 upright fluorescent microscope using a ×40 (0.55 NA) water-immersion objective (Nikon). Dissected preparations were
maintained in crayfish saline [modified Van Harreveld's solution (in
mM): 205 NaCl, 5.3 KCl, 13.5 CaCl2, 2.45 MgCl2, and 0.5 HEPES/NaOH, pH 7.4] at 14°C.
The entire opener muscle is innervated by a single tonic excitatory and
inhibitory motor neuron (Cooper et al. 1995a
). To
visualize the nerve terminals, living preparations were stained
fluorescently for 2-5 min. with 2-5 µM 4-[4-(diethylamino)
styryl]-N-methylpyridinium iodide (4-Di-2-Asp; Molecular
Probes, Eugene, OR) in crayfish saline.
Protein microinjection
Immediately before injection the proteins were mixed with a
Texas Red dextran solution (70 kDa, Molecular Probes) to a final concentration of 0.51 mg/ml protein and 0.05% of Texas Red dextran. This solution then was loaded into the microelectrode by capillary action from the back of the electrode until the tip of the electrode was filled sufficiently so that the wire from the electrode holder touched the solution interface. The microelectrode then was placed into
the excitatory axon of the opener muscle close to the axon bifurcation.
Within 30 min of the start of pressure injections, the 70-kDa Texas Red
dextran had entered nerve terminals and loaded the varicosities (Fig.
1C). Because the molecular weights of -SNAP and
-SNAP(L294A) mutant are 35 kDa, it would be expected that they would
have also diffused into the varicosities during this time. The membrane
potential of the axon was in the range of
60 to
70 mV with this
type of solution loaded into the electrode. To inject the protein,
pressure was applied with a PicoSpritzer II (General Value, Fairfield,
NJ) within a pressure range of 10-60 psi. The pressure was varied
depending on how well the axon filled. Because the excitatory motor
axon is stimulated selectively in the meropodite, only it will produce
action potentials; therefore it is possible to confirm penetration of
the excitatory axon by the microelectrode.
Evoked postsynaptic potential measurements
Intracellular muscle recordings were made with a 3 M
KCl-containing microelectrode placed in a centrally located fiber in the opener muscle (Fig. 2, A
and B). The responses were amplified with a 1 × LU
head stage and an Axoclamp 2A amplifier (Axon Instruments, Foster City,
CA). Axons were stimulated by a train of 10 pulses given at the
indicated frequencies. The stimulation frequency was kept constant for
each preparation. The range in frequencies used varied from 40 to 60 Hz
with a train interval of 10 s. The frequency chosen was determined
at the time of the experiment to ensure an excitatory postsynaptic
potential (EPSP) response by the fourth pulse of the train. Because the
stimulus train is phase locked, one readily can measure each of the 10 peak amplitudes of the EPSPs and the sweep number in which they
occurred. The sweep number, the mean value of the baseline, and the
maximum of EPSP amplitudes were all recorded. All events were measured and calibrated with the MacLab Scope software 3.5.4 version
(ADInstruments, Mountain View, CA). Stimulation was obtained using a
Grass S-88 stimulator and a stimulation isolation unit (Grass
Instruments, Warwick RI) with leads to a standard suction electrode
(Cooper et al. 1995b).
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To quantitatively compare the changes caused by the injected agents in
the various preparations, the measurements were normalized to a percent
change. The percent change from baseline was calculated using the
difference among the average of the first 100 EPSP events during the
baseline recording before injection and the average of the 100 EPSP
events around the maximum response during the injection procedure and
then dividing the result by the baseline value as shown in the
following equation: [(Baseline maximum response)/Baseline] × 100% = % Change. The means of the calculated percent changes from
baseline among preparations in which the carrier buffer only was
injected into the axon, the
-SNAP and the
-SNAP(L294A) mutant
were graphed for comparative purposes (Fig. 2C).
The 10th EPSP peaks of a train were used to calculate a facilitation
index (Fe) with respect to the earlier EPSP amplitudes (Crider and Cooper 1999). To calculate facilitation
indices, the amplitude of the 10th EPSP was divided by one of the
previous EPSP amplitudes and the result was subtracted from one. A
facilitation index for each pulse in the train (X) by the
equation Fe(10/X) = (10th
EPSP/Xth EPSP)
1. The ratios were calculated for
events 1-9 in each trial.
Excitatory postsynaptic current measurements
Focal macropatch recording was used to measure synaptic
currents. The synaptic currents were obtained using the loose patch technique by lightly placing a 10- to 20-µm diam, fire-polished, glass electrode directly over a single, spatially isolated varicosity along the vital dye-visualized nerve terminal. The macropatch electrode
is specific for current recordings within the region of the electrode
lumen. The lumen of the patch electrode was filled with the same
solution as the bathing medium, and the seal resistance was in the
range of 100 k to 1 M
. Because the seal can be lost easily if the
muscle twitches under the electrode, stimulation was restricted to a
range of 1-2.5 Hz. Evoked EPSCs (excitatory postsynaptic currents) and
mEPSCs (miniature EPSCs) were recorded and analyzed to determine the
mean quantal content (m), the number of release sites
(n), and the average probability of release at a terminal
(p) (Cooper et al. 1995b
, 1996b
). In each
synaptic current recording, a trigger artifact and a nerve spike can be visualized that indicates nerve stimulation. Mean quantal content can
be determined by direct counts (mco):
direct counts (mco) = [
(failures × 0), (single events × 1),
(double events × 2) etc.]/total number of sweeps.
As shown in Fig. 3A, in some cases there were no evoked events that follow the nerve terminal spikes. This type of response is called a failure in evoked release, and is given the value of zero. If only one single event occurs after the spike, it is counted as a single-evoked event and is given the value of one (Fig. 3B). When double events occur, they are referred to as double-evoked events and are counted as two (Fig. 3C), etc.
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The determination of quantal release over the time of the
experiment is made possible by examining the area of the evoked current
which is a measure of charge (Fig. 4). It
should be noted that the time of peak evoked events varies due to
latency jitter so the measurements of peak amplitude are not as
reliable as the charge measure when multiple events occur (Fig.
3C). The change in the charge measures before and after
loading proteins in the nerve terminals and the histograms of the
frequency in occurrence of charge of the evoked events were calculated.
The data sets were tested for a best-fit approximation based on
assumptions discussed in earlier reports (Cooper et al.
1995b; Wernig 1975
). Binomial
distributions are known to represent the quantal nature of release in
crayfish neuromuscular junctions (Wojtowicz et al. 1991
). To test for nonuniform binomial distributions, the
procedures described earlier were used (Wojtowicz et al.
1991
). The
2 statistic and a modified Akaike
information criterion (AIC) were used to estimate the distribution that
best fits the observed distribution of events. Because the injection of
the protein produced gradual changes in all the quantal parameters,
sample sets of data for every 400 events were used and were found to be
sufficient to obtain statistically significant values for quantal
predictions.
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Analysis of latency
Latency was measured as the time period between the starting
point of the spike (extracellular recorded action potential) and
starting point of evoked events (for the 1st and the 2nd events) (Fig.
6, A and B). A representative plot of the
frequency of occurrence at various latencies is provided (Fig.
7A), and a normalized graph for the occurrence of events is
shown for the two curves in Fig. 7B, as plotted in
cumulative frequency for the latencies measured (i.e.,
Kolmolgrov-Smirnov) (Zar 1999).
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RESULTS |
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Presence of -SNAP in crayfish neurons
Western blotting experiments confirmed the presence of -SNAP in
crayfish neurons. Not surprisingly, the anti-bovine
-SNAP antibody
was able to cross-react with the crayfish SNAP. This is consistent with
the high degree of sequence conservation between
-SNAPs identified
from a wide variety of species (e.g., Drosophila and bovine
-SNAP are 61% identical) (Pallanck et al. 1995
). The crayfish protein had a similar molecular weight to that of the rat
protein (Fig. 1A), and comparable levels of protein were
seen in rat brain and crayfish CNS. Immunofluorescence studies indicate that crayfish
-SNAP is present in both excitatory and inhibitory motor neurons and appears concentrated in the nerve terminals. This is
born out by the staining of the varicosities in Fig. 1B. Extensor muscles containing both phasic and tonic excitatory motor neurons also show neuronal staining with this antibody (data not shown). From these data, it appears that
-SNAP is concentrated in
the terminals of each of the two classes of motor neurons that innervate the crayfish muscle (Fig. 1C).
Effects of -SNAP on EPSP amplitudes
Measuring the change in EPSP amplitude readily can provide an
indication of whether a microinjected protein alters NT release. To
test the effect of -SNAP during injection, the axon was stimulated by a train of 10 pulses at 40 Hz with an interval of 10 s, and 10 EPSP responses were recorded by an intracellular electrode. As shown in
Fig. 2A,
-SNAP enhanced the amplitudes of the EPSPs. The
superimposed graphs of evoked EPSPs for the control and
-SNAP injected axons show that there is a consistent increase in the amplitudes. This enhancement of EPSP amplitude was most obvious at
later stimulus events in the trains. For this reason, the 10th responses from each train were recorded verses time and plotted in Fig.
2B. (Although clearly not a linear function, a best-fit line
was drawn through each data set to aid in comparisons.) Amplitude increases were seen only after the dye/
-SNAP solution began to appear in the nerve terminal (denoted by the vertical line on Fig.
2B). The
-SNAP(L294A) mutant had the opposite effect,
causing a decrease in the EPSP amplitudes (Fig. 2B). This
decrease initiated after the dye/protein entered the nerve terminals.
The sham injections resulted in slight increase in the amplitudes of
the EPSPs. This indicates that the injection process may have a
positive effect on EPSP amplitude, suggesting that the effect of
-SNAP may be slightly overestimated, whereas the inhibition by
-SNAP(L294A) was slightly underestimated.
The changes in the EPSP amplitudes were normalized to a percent change,
which was determined by the difference of the average in the first 100 EPSPs events during the baseline and the average in the 100 EPSPs
events around the maximum response (Fig. 2C). Although there
was some variability in the percent change from preparation to
preparation, it is clear that -SNAP causes an almost twofold
increase in EPSP amplitude. The dominant negative mutant, however, was
clearly inhibitory leading to a 20% decrease in the 10th EPSP peak
amplitude. For the purpose of illustration, data obtained using the
10th EPSP are shown in Fig. 2C, yet in most preparations,
the maximal effect of the injected proteins occurred between the 3rd
and the 5th EPSP peaks of the stimulus train. When the 4th peaks were
compared with sham injected, there was a two- to fourfold increase in
EPSPs (n = 3) when
-SNAP was injected and a 45-80%
decrease in EPSPs (n = 3) when the mutant
-SNAP was
injected (data not shown). The fact that
-SNAP has a positive effect
on EPSP amplitude and the dominant negative
-SNAP(L294A) mutant has
an inhibitory effect suggests that the mammalian proteins are
functional in the crayfish NMJ. This justifies the use of these
proteins to study the role of
-SNAP in NT release at these NMJs.
-SNAP does not affect facilitation of EPSPs
Facilitation, or the progressive increase of EPSP amplitude during
the course of a train of stimuli, is thought to be indicative of a
progressive increase in calcium concentration in the nerve terminal
(Bain and Quastel 1992; Delaney et al.
1989
). By measuring the effect of a microinjected protein on
facilitation, one can assess the calcium requirement for that protein
to act. The EPSP amplitudes for each event within a train were recorded
during the entire injection period. A facilitation index for each pulse (X) in the train was calculated by the equation
Fe(10/X) = (10th
EPSP/Xth EPSP)
1. As an example of this type of
analysis, Fe(10/1) is plotted versus
injection period time for the three different treatments (Fig.
2D). There was no change in
Fe(10/X) in any of the
injection experiments when the numerous possible Fes were
calculated. This indicates that EPSP amplitudes from the 1st event to
the 10th event increased proportionally. These data demonstrate that
neither
-SNAP nor the mutant affect facilitation of NT release in
the crayfish NMJ, suggesting that the role of
-SNAP is not calcium dependent.
-SNAP effects on quantal release
-SNAP increases the EPSP amplitudes of crayfish NMJ,
consistent with its effect on squid axons described by DeBello
et al. (1995)
. Taking advantage of the crayfish system, we
turned to quantal analysis to determine the potential mechanisms by
which
-SNAP causes this increase. The crayfish NMJ is ideal for this type of analysis because its relatively low output allows single events
to be monitored and statistical analysis to be applied. The number of
evoked events and failures before (Zone I in Fig. 4) and during
-SNAP injections (Zone II and III in Fig. 4) was counted directly,
making sure to distinguish single events from multiple events (as
delineated in Fig. 3). The evoked current over time (charge) was used
to assess synaptic release and spontaneous events. The charge measure
is a more appropriate measure than peak amplitudes because of the
latency jitter in evoked release, and it allows the rates of release to
be determined. The evoked charge was enhanced as
-SNAP began to
appear under the electrode (Fig. 4A, compare Zone I with II
and III). Once the dye filled the nerve terminals, more evoked events
were obtained and there was an increase in the charge of these events
(Fig. 4A). The increase in evoked charge, after
-SNAP
reached the terminals, is indicated by the rightward shift in the
histogram distribution of the data set shown in Fig. 4B.
Larger evoked charges and fewer failures were present after the protein
was well loaded in the terminal (Fig. 4B, zone III).
-SNAP affects not only the evoked charge but also the occurrences of
evoked events and the number of failures. The numbers of failures
decreased after the dye began to reach nerve terminals and were
maintained at low levels throughout the injection period (Fig.
5A). The rate at which the
failures occurred also decreased as the dye reached the terminals. The
reduced failure rate stayed constant for the remainder of the
experiment (Fig. 5B). This dampening of failure rate was
common in the six preparations in which
-SNAP was injected.
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Mean quantal content (mco) is the
average number of evoked-release events per action potential. As shown
in Table 1 for one experiment, -SNAP
injection leads to an increase in quantal content. This increase was
seen in all six experiments, ranging from +7 to +124% relative to the
uninjected control (Table 2). An increase in quantal content could be due to an increase in the number of release
sites or to an increase in the number of release events per site. To
distinguish between these two possibilities, the total number of
failures, single- and multiple-evoked events in 400 sweeps were
combined, and the best fit of the data set distribution was calculated.
From this analysis, the quantal parameters, n (number of
release sites) and p (probability of release at a site), can
be determined by the methods previously described (Cooper et al.
1995b
). In Table 1, the p values represent an
average value. Typically, injection of
-SNAP did not significantly
change n, relative to sham injection, in any of the six
preparations. However,
-SNAP injection consistently increased
p ranging from +27 to +161%, relative to control (Table 2).
These data indicate that
-SNAP increases the probability of release
at a site but does not increase the number of release sites.
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Effect of -SNAP on latency
Latency is defined as the time required for an action potential to
induce NT release. In molecular terms, it is the time required for a
primed and docked SV to respond to the calcium influx and fuse with the
active zone. Measuring from the starting point of the spike to the
starting point of an evoked event in the EPSC records, it is possible
to calculate the time needed for an evoked event to occur (Fig.
6). In the crayfish NMJ, the fastest
events took ~1-2 ms. After injection of -SNAP the fastest events
stayed the same but more of these fast events occurred. The
superimposed histograms in Fig.
7A indicate that more events
had shorter latencies after
-SNAP was loaded into the terminals.
Synaptic vesicular fusion rates became apparently faster because more
vesicles were "releasable" in the presence of
-SNAP, thus the
larger amplitude bins in the histograms (Fig. 7A). Because
there are more events occurring, it is hard to visualize if there is a
leftward shift in the histogram or if the difference is just due to
more events. Because of this ambiguity, the latency was normalized into
cumulative frequency plots (Fig. 7B). From this analysis of
the data, it becomes more obvious that there is a slight leftward shift
(decrease in latency) as more events occur at an earlier time
throughout the normalized distribution. Presumably the larger number of
releases indicates that more quanta are releasable. However, shift in
the histogram would simply indicate that slower releases are sped up.
These "slowly released" quanta are releasable, but they might be
released more rapidly in the presence of
-SNAP. This trend was seen
in six different preparations injected with
-SNAP.
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DISCUSSION |
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In summary, the results presented here suggest that the role of
-SNAP in the synapse is to increase the pool of "readily releasable" synaptic vesicles. This is supported by several
observations. Initially, microinjection of
-SNAP caused an increase
in synaptic output (Fig. 2) and NT release could be inhibited by the
dominant negative
-SNAP(L294A) mutant. Neither protein had an effect
on facilitation (Fig. 2D), indicating that
-SNAP did not
alter calcium buffering or loading within the terminal, and thus
calcium channels were unaffected by the protein injections. Quantal
analysis of the effects of microinjected
-SNAP showed that it caused
a decrease in the failure rate of NT release (Figs. 4 and 5) and led to
a slight decrease in release latency (Fig. 7).
-SNAP did not
accelerate the minimal process required for SV fusion but increased the
availability of vesicle for release at the maximum rates (Fig.
7B). These findings suggest that
-SNAP causes an increase
in the fusion competence of SVs without significantly effecting NT
release kinetics. This is supported further by statistical analysis of
the quantal data (Tables 1 and 2). From this analysis, it is clear that
-SNAP serves to increase the probability (P) that a
stimulus will result in a release event but it does not significantly
increase the number of sites (n) at which release can occur.
Mechanistically, these data support the model in which SNAPs (and
perhaps by extension NSF) are involved in increasing the pool of fusion
competent SVs but are not involved in the actual membrane fusion
events. Because transmitter release was inhibited by the dominant
negative
-SNAP(L294A) mutant, it appears that the mutant competed
for interaction of the native
-SNAP, which is likely present in much
lower amounts than the injected mutant
-SNAP. The effects were
gradual and increased with injection time, suggesting a selective
interaction and not a massive nondirected protein interaction on ionic
channels or on other aspects of the release machinery.
The low output tonic motor nerve terminals of the crayfish makes it an effective preparation to address questions related to the kinetics and mechanics of synaptic transmission because latency measurements and quantal analysis can be performed readily. This type of analysis, available with the crayfish NMJ, is not feasible in higher output synapses such as the squid giant synapse and the Drosophila NMJ or among vertebrate neurons in culture. The low output of tonic motor nerve terminals is partly due to the synaptic ultrastructure because these synapses may contain one or a few active zones where evoked release can occur. This architecture results in a relatively simple synaptic structure to investigate alterations in the number of vesicles docked around an active zone by electron microscopy studies. Likewise physiological measures of small changes in synaptic efficacy can be quantified to assess manipulations of the release machinery.
We addressed potential mechanisms in -SNAP function by examining the
quantal parameters and by determining if the timing of evoked events is
altered. This tonic low-output motor nerve terminal is ideal for this
study because it normally shows latency jitter as indicated by the
range of times between depolarization and vesicle release. By measuring
the latencies of the first evoked events before and during
-SNAP
injections, one can determine if the mechanics of vesicle
docking/priming are altered. Because the number of events also
increases along with more events occurring with a shorter latency,
histograms of the occurrence of events at various times (Fig.
7A) is not of particular use. The relative cumulative graphs
of latency are more beneficial for determining shifts in the latency
but the shifts only can be observed within data sets because the
cumulative graphs are first-rank summed to measure relative differences
(Fig. 7B). Groups of 400 evoked trials were compared before
and during injections of
-SNAP. After the terminals began to be
loaded with
-SNAP, no further shifts were detected among the groups
of data sets. The shifting of the vesicle pool to minimum release times
reveals that an increase in
-SNAP at the terminal promotes the
fusion competence of vesicles but does not effect the time required for
the Ca2+-influx-induced membrane fusion process,
which is consistent with earlier work in neurons (DeBello et al.
1995
) and endocrine cells (Kibble et al. 1996
;
Martin et al. 1995
). Only ultrastructural analysis after
rapid fixation and subsequent serial sectioning of the synapses would
allow one to prove such a postulation anatomically. The physiological
data suggest that
-SNAP is working to enhance vesicle
docking/priming. With the methods used in this study, we are not able
to assess whether
-SNAP also plays a role in vesicle recycling as
might be inferred from the demonstration of a role for NSF in this
process (Littleton et al. 1998
).
Several aspects can contribute to the diversity of NT release by nerve
terminals. Ultrastructural analysis of nerve terminals suggest that
there is great deal of heterogeneity in the number of vesicles that can
encircle the active zones of the synapses (Govind et al.
1994). This suggests that various number of vesicles could be
primed for release in each active zone; however, the conformational
constraints of the active zone suggest that there may be a limit to the
total number of primed/docked vesicles possible. Also, depending on the
synaptic complexity (Cooper et al. 1996b
,c
; King
et al. 1996
; Msghina et al. 1998
), multiple
active zones may be present on a single synapse, thus providing a
structural basis for synaptic differentiation to effect synaptic
efficacy. These structural arrangements of active zones could allow for a fine-tuning of neurotransmission by specific neurons. Additionally, cytoplasmic proteins such as
-SNAP could provide a mechanism for
fine-tuning synaptic efficacy at the established active zones. This
could be accomplished by regulating the level of expression or of
axonal transport of these proteins. Because microinjected
-SNAP does
increase NT release in both squid axons (DeBello et al.
1995
) and in crayfish NMJs, it seems simple up- or
downregulation of SNAP levels at the synapse could be used to modulate
NT release. Alternatively, there are other plausible mechanisms that
could facilitate vesicle docking/priming to control the efficacy of NT
release. The effect of stimulation on protein phosphorylation events is
a viable mechanism because it is well known that the entry of calcium
can lead to the activation of various protein kinases such as protein
kinase C and calmodulin kinase II (Makhinson et al.
1999
; Valtorta et al. 1996
). For example,
phosphorylation of synapsins by the calcium-activated kinases has been
shown to increase the pool of free SVs by facilitating their release
from the presynaptic cytoskeleton (Benfenati et al.
1992
). Several other studies have shown the elements of the
synaptic secretory machinery can be phosphorylated in vitro [e.g.,
Munc18/nSec1 (Fujita et al. 1996
; Shuang et al.
1998
), SNAP-25 (Risinger and Bennett 1999
;
Shimazaki et al. 1996
), synaptophysin (Barnekow
et al. 1990
; Rubenstein et al. 1993
), and
synaptotagmin (Bennett et al. 1993
; Davletov et
al. 1993
; Popoli 1993
)] and that
phosphorylation affects their interactions with other secretory
machinery components. Although all of these are possible mechanisms to
control synaptic efficiency, detailed experiments are needed to address
their relative importance in causing enhanced synaptic release during
short- and long-term synaptic facilitation (Delaney et al.
1991
; Dixon and Atwood 1989a
,b
; Dudel et
al. 1983
; Parnas et al. 1982
; Zucker and
Fogelson 1986
) and among terminals that show differences in synaptic efficacy (Atwood and Cooper 1995
, 1996a
,b
;
Atwood and Wojtowicz 1986
; Atwood et al.
1994
; Bradacs et al. 1997
; Cooper et al.
1998
; King et al. 1996
; LaFramboise et
al. 1999
).
The stochastically derived quantal parameters n and
p, are to help one determine possible mechanisms but because
the calculated n is difficult to directly correlate to a
structural identity, it remains open for debate if much weight can be
placed on such indices for a structural meaning. Latency, facilitation
measures, and direct quantal counts for the calculation of m
are not as subjective indices as n and p, and
thus are emphasized in this report. It is beyond the scope of this
report to dwell on the finer points of quantal analysis but to
highlight a few points may clarify the reason of not placing to much
emphasis on n and p for mechanistic
interpretations here or in future descriptions. The synaptic structure
of the crayfish neuromuscular junction is one in which multiple
vesicles can dock around an dense body (i.e., an active zone); in
addition, a synapse can have multiple active zones with varied spacing
between them (Cooper et al. 1995a, 1996c
). So the
problem with a structural interpretation of n is that
n maybe inferred to be the various numbers of docking sites around a single active zone or each active zone itself. In vertebrate synaptology, n is interpreted to represent an entire bouton
or varicosity that contains multiple synapses with each synapse
containing multiple active zones (Korn et al. 1981
). At
least with the simplest of the crustacean synapses and the structural
reconstructions of recorded varicosities (Atwood and Cooper
1996a
,b
; Atwood et al. 1994
; Cooper et
al. 1995a
, 1996c
), an anatomic meaning for n may be
forthcoming when better stochastic analysis can be performed when
synaptic efficacy is altered experimentally. For now, if one allows
n to represent a single active zone, injection of
-SNAP would likely enhance the number of docked vesicles within each single
active zone, thus increasing the probability that each would release a
single quantum. But if the same active zones are being used, then only
p will increase and n would remain constant. If
additional active zones were recruited at the same stimulation frequency used for the analysis, then n would increase. In
that case, however, it is likely that p, which represents
the average probability of release, would decrease. Because the
p values reported are average p's, when
n increases p may decrease, unless of course p increases to such an extent that it is still larger after
the division of n. The fact that we measured an increase in
p with the stimulation paradigm used suggests the same
active zones were used with an enhanced probability of release. These
active zones are likely the ones in close concert with neighboring
active zones on a single synapse (see Cooper et al.
1996c
for the computational interpretations). One would predict
that n would increase with an increase in the presence of
-SNAP, and this likely may be seen when quantal assessment can be
performed with accuracy at higher stimulation frequencies, but at
present we do not feel that the analysis can accurately be performed
when n is >3 (personal conversation with Dr. Bruce Smith
developer of the quantal analysis software used, Dalhousie University,
Canada), and therefore we did not assess substantially higher
stimulation frequencies while increasing the amount of
-SNAP within
the terminals.
What is clearly demonstrated in this paper is that -SNAP does
increase synaptic output by increasing the probability that a stimulus
will result in NT release. On cellular terms, this suggests that
-SNAP increases the ready release pool of fusion competent SVs. On a
molecular basis, we must assume that
-SNAP is effecting NT release
by acting on the SNARE proteins present in the active zone and SVs,
though that was not specifically addressed here. This would imply that
-SNAP functions not in the fusion event but before calcium influxes
to induce an activated conformation in the SNARE proteins so that they
are primed to participate in membrane fusion.
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ACKNOWLEDGMENTS |
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Funding for this research was provided by the University of Kentucky Research and Graduate Studies Office (R. L. Cooper), National Science Foundation Grant IBN-9808631 (R. L. Cooper) and NSF-ILI-DUE 9850907 (R. L. Cooper) as well as an undergraduate training fellowship from Howard Hughes Medical Institute (R. C. Southard), and National Heart Lung, and Blood Institute Grant HL-56652 (S. W. Whiteheart).
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FOOTNOTES |
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Address for reprint requests: R. L. Cooper, T. H. Morgan School of Biological Sciences, 101 Morgan Bldg., University of Kentucky, Lexington, KY 40506-0225.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 June 1999; accepted in final form 2 September 1999.
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REFERENCES |
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