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The Journal of Neuroscience, October 1, 1999, 19(19):8419-8434
Calcium Entry Related to Active Zones and Differences in
Transmitter Release at Phasic and Tonic Synapses
M.
Msghina1,
A. G.
Millar1,
M. P.
Charlton1,
C. K.
Govind2, and
H. L.
Atwood1
1 Department of Physiology, Medical Research Council
Neural Group, University of Toronto, Toronto, Ontario, Canada M5S 1A8,
and 2 Life Sciences Division, University of Toronto at
Scarborough, Toronto, Ontario, Canada M1C 1A4
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ABSTRACT |
Synaptic functional differentiation of crayfish phasic and tonic
motor neurons is large. For one impulse, quantal release of
neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes
Calcium Green-1 and fura-2. Estimated Ca2+
entry increased almost linearly with frequency for both axons and was
two to three times larger in phasic terminals. Tonic terminal Ca2+ at 10 Hz exceeded phasic terminal
Ca2+ at 1 Hz, yet transmitter release was much
higher for phasic terminals at these frequencies. Freeze-fracture
images of synapses revealed on average similar numbers of prominent
presynaptic active zone particles (putative ion channels) for both
neurons and a two- to fourfold phasic/tonic ratio of active zones per
terminal volume. This can account for the larger calcium signals seen
in phasic terminals. Thus, differences in synaptic strength are less
closely linked to differences in synaptic channel properties and
calcium entry than to differences in calcium sensitivity of transmitter release.
Key words:
crustacea; crayfish; synaptic differentiation; tonic; phasic; ultrastructure; active zone; focal recording; calcium imaging; quantal release
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INTRODUCTION |
Synapses display a wide range of
functional properties. Some show a high probability of neurotransmitter
release by the nerve impulse, whereas others have a low probability of
release. As yet, there is not a consensus of opinion on the important
factors determining synaptic efficacy (Walmsley et al., 1998
), although several mechanisms have been proposed (Parnas et al., 1982; Cooper et
al., 1995a; Harris and Sultan, 1995; Dobrunz and Stevens, 1997; Murthy
et al., 1997; Schikorski and Stevens, 1997). In this study, we tested
one possibility: synapses that release a relatively large amount of
transmitter admit more calcium than synapses that release relatively
little transmitter, and the larger calcium entry accounts for the
larger output of transmitter.
Motor neurons of crustaceans provide excellent models for the study of
synaptic differentiation. Individual junctions of a single neuron on
different target muscle fibers produce EPSPs varying greatly in
amplitude and facilitation (Atwood, 1967; Bittner, 1968; Atwood and
Bittner, 1971; Cooper et al., 1995a). Even greater differences in
transmitter output occur at junctions of the physiologically distinct
"phasic" and "tonic" motor neurons (Kennedy and Takeda, 1965a,b; Atwood, 1976; Atwood and Wojtowicz, 1986). Phasic EPSPs are
usually much larger than tonic EPSPs, even in the same target muscle
fiber (Bradacs et al., 1997), and typically show depression rather than
facilitation during sustained stimulation (Atwood, 1976; Lnenicka,
1991). Differences in transmitter output account for these features
(Atwood and Wojtowicz, 1986). Parallel findings have been shown in
amphibian and other neuromuscular junctions (Pawson and Grinnell, 1984;
Wernig et al., 1984; Herrera et al., 1985; Rash et al., 1988), as well
as in the central nervous systems of several species (Davis and
Murphey, 1994; Stevens and Wang, 1995). Thus, patterns and principles
of synaptic operation found in crustacean synapses are shared with
other synapses, including mammalian central synapses.
An obvious explanation for differences in transmitter output at phasic
and tonic synapses is that calcium entry is greater at phasic synapses,
either because there are more calcium channels or because the calcium
channels have a higher probability of opening and admit more calcium
per nerve impulse. To examine this possibility, we compared the quantal
release and calcium signals of phasic and tonic terminals at several
frequencies of stimulation. Morphological data were obtained from
images of synaptic active zones in freeze-fracture replicas. In these
images, prominent membrane particles that are thought to represent the
synaptic calcium channels can be seen (Pumplin et al., 1981; Walrond
and Reese, 1985; Haydon et al., 1994). Our estimates of the relative
effectiveness of individual active zones in admitting calcium showed
that, on average, active zones of phasic synapses are not more
effective than those of tonic synapses in admitting calcium during a
nerve impulse. When frequency of stimulation of the tonic axon is
raised to increase calcium entry, quantal output remains less, although
calcium accumulation is greater, than for the phasic terminals
activated at a much lower frequency. Thus, contrary to the initial
hypothesis, relative calcium entry at active zones is not the main
factor responsible for the large differences in transmitter release at
phasic and tonic synapses.
Preliminary results have been published previously in abstract form
(Msghina et al., 1995).
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MATERIALS AND METHODS |
Animals and preparation. Freshwater crayfish
(Procambarus clarkii; 2-4 cm body length) were obtained
from the Atchafalaya Biological Supply Company (Baton Rouge, LA)
and maintained under standard laboratory conditions (Bradacs et al.,
1997). The extensor of the carpopodite (main leg extensor muscle),
innervated by two excitatory motor axons, one phasic and one tonic, was
selected for experimentation. The general anatomical and physiological features of the extensor muscle, and its preparation, have been described previously (Bradacs et al., 1997; Msghina and Atwood, 1997).
The preparation was dissected and maintained in a modified Van
Harreveld's crayfish solution containing (in
mM): NaCl 205.3, KCl 5.3, CaCl2.2H2O 13.5, MgCl2.6H2O 2.5, and HEPES
buffer at pH 7.5). To minimize contractions, the muscle was stretched
after cutting the membrane connecting the meropodite and carpopodite, leaving the tendon attachment intact. Most of the experiments were
performed at room temperature (19-20°C); however, focal
"macropatch" recordings were performed at 13-14°C to aid the
counting of quanta (see below).
Electrophysiological recording. Properties of
neurotransmitter release were assayed by extracellular recording at
visualized phasic and tonic nerve endings. The procedures were similar
to those described for this preparation in Msghina et al. (1998). Focal
macropatch recording (Dudel, 1981; Wojtowicz et al., 1994; Cooper et
al., 1995a) of the prejunctional nerve terminal spike and excitatory
junction current (EJC) from single tonic and phasic terminals was
performed after exposing the preparations to the vital dye
4-(4-diethylaminostyryl)-N-methylpyridinium iodide
(4-Di-2-Asp, Molecular Probes, Eugene, OR) for 3 min at a concentration
of 2 µM to visualize the terminals (Cooper et
al., 1995a). The macropatch electrode, pulled from Kimax glass (outer
diameter, 1.5 mm), was fire-polished to an internal diameter of 15-20
µm, and the tip was bent to fit under a 40× Nikon water immersion
objective lens (working distance, 1.0 mm). The electrode, filled with
the standard physiological solution, was placed under visual control
over a well defined tonic terminal together with a closely adjacent
phasic terminal (see Fig. 2A), so that both tonic and
phasic responses were recorded from the same patch of muscle, thus
circumventing the need to compensate for differences in seal or
electrode resistance in comparing the responses. The patch-clamp
amplifier was obtained from Zeitz-Instrument Vertriebs (Augsburg,
Germany). Seal resistance was monitored throughout the recording by
passing test pulses through the electrode.
The excitatory axons were stimulated by pulses delivered through a
suction electrode into which the cut ends of both the tonic and phasic
axons were drawn. The tonic axon, which is larger in diameter and has a
lower threshold of activation (Bradacs et al., 1997), was first
selectively excited by adjusting the amplitude and duration of the
applied voltage, and stimulated with 500-1000 pulses at frequencies of
1-20 Hz. After this, the stimulation intensity was increased to
recruit the phasic axon, which was stimulated with 500-1000 pulses at
frequencies of 1-5 Hz. The electrophysiological data were collected
and analyzed by means of a computer-based data acquisition system. The
procedures for estimating quantal content of the evoked responses at
different frequencies, and the fitting of the data to Poisson or
binomial distributions, have been described previously (Wojtowicz et
al., 1991, 1994; Cooper et al., 1995a,b; Msghina et al., 1998).
Calcium indicators and imaging. Two calcium
indicators, Calcium Green-1 and fura-2, were used in the experiments to
observe intracellular calcium in nerve terminals. Calcium Green-1 was selected for its ability to produce measurable changes in calcium signal at low frequencies of stimulation (Cooper et al., 1995a), and
initial experiments were performed using this indicator. The dye (1 mM, in 100 mM KCl), was injected into either
the tonic or phasic axon with a sharp microelectrode (resistance,
50-70 M
in 100 mM KCl), using a Picospritzer II
pressure-injection apparatus. Continuous pressure was applied at 20-40
psi for 5-15 min. The two axons are nearly the same diameter (Bradacs
et al., 1997), and when they had been injected for the same period of time with the same electrode, they had similar concentrations of the
dye, as judged from fluorescence of the major axon branches. After a
rest period of 30 min to allow adequate diffusion of the dye into the
terminals, the preparations were stimulated for 8 sec at each of the
following frequencies: 1, 2, 5, 10, 20, 30, and 50 Hz. The terminals
were visualized using a Nikon 40× water immersion lens, and frames
were captured by a confocal microscope at 1 frame/sec (see below). To
ascertain that the excitatory axons were activated throughout the
stimulation, the action potential was also monitored continuously.
Images for the Calcium Green-1 experiments were acquired, stored, and
analyzed by a Bio-Rad 600 confocal laser scanning microscope (Bio-Rad,
Hercules, CA) and associated software. Images included the entire
thickness of each sampled bouton. Calcium Green-1 fluorescence was
observed during illumination by the 488 nm line of a 25 mW argon laser,
attenuated to 1% of maximum with neutral density filters. Fluorescence
emission was detected with a low-pass filter (cutoff at 514 nm).
Background fluorescence was subtracted from resting and
stimulation-induced fluorescence intensity values. At each frequency,
the resting fluorescence value was subtracted from the
stimulation-induced value, and the result (
F) was
normalized to the resting fluorescence intensity (F)
to give F/F.
During stimulation, the normalized fluorescence values for Calcium
Green-1 (F/F) provided the initial
measure of calcium change in the terminals. The dependence of these
values on intraterminal calcium concentration was determined by
measuring the fluorescence in calibration solutions with known
[Ca2+] (Fig.
1). These solutions, obtained from
Molecular Probes (Calcium Indicator Calibration Kit) were imaged in 50 µm cuvettes (Vitro Dynamics, Rockaway, NJ) to determine how close
experimentally predicted concentrations were to actual values. First,
Fmax (fluorescence intensity at
saturating [Ca2+]) and
Fmin (fluorescence intensity at zero
[Ca2+]) were determined in
vitro for the selected dye concentration (10 µM Calcium Green-1). The
Kd value for Calcium Green-1 was estimated to be 138 nM, from the linear plot of
log(F 
Fmin)/(Fmax F) against
log[Ca2+] (Fig. 1A).
Next, new solutions with varying levels of
Ca2+ and the same dye concentration were
imaged, and the fluorescence intensity was recorded. Measured
fluorescence values were inserted into the equation
[Ca2+] = Kd(F Fmin)/Fmax F) to obtain predicted
[Ca2+] concentrations for a given
fluorescence intensity. The predicted concentrations were then compared
with the "actual" concentration of the calibration solution
reported by Molecular Probes. The fluorescence of Calcium Green-1
accurately predicted calcium concentrations to ±10% if the dye
concentration was kept constant (Fig. 1C). When
F/Fmin was plotted
against log[Ca2+], the relationship was
linear over the range 2 × 10
8 to
106
M (Fig. 1B); at higher
concentrations, the slope of the curve decreased and thus the readings
above 106 M
were less accurate. From these calibrations, and with the resting values of Ca2+ determined from fura-2
measurements, the change in fluorescence of Calcium Green-1 was used as
a measure of the change in [Ca2+] when
the latter value was below
106
M.
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Figure 1.
Calibration curves for Calcium Green-1.
A, Relationship between fluorescence measurements and
free calcium, with an estimate of Kd.
B, Relationship between normalized fluorescence change
and free calcium, showing a linear relationship between normalized
fluorescence and log[Ca2+] between
108 and
106 M
[Ca2+]. C, Predictive value of
Calcium Green-1. Using predetermined values for
Rmax,
Rmin, and
Kd, Calcium Green-1 fluorescence
values (F) from several calibration solutions
(Molecular Probes Calcium Imaging Calibration Kit) were substituted
into the equation [Ca2+] = Kd(F Fmin/Fmax F). The predicted value of
[Ca2+] given by the equation is plotted against
the actual value reported by Molecular Probes. The shape of this
relationship is linear, with a slope of ~1, indicating that Calcium
Green-1 accurately predicts [Ca2+] in the range
plotted. F, Fluorescence at × [Ca2+]free;
Fmax, fluorescence at saturating
[Ca2+]free;
Fmin, fluorescence at 0 [Ca2+]free,
F = F Fmin.
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Measurements of resting and stimulus-evoked
[Ca2+] were also made with the
ratiometric calcium indicator fura-2. Estimation of
[Ca2+]i was more
direct using the ratiometric dye, and the kinetics of rise and decay of
[Ca2+]i could be
better resolved because of the imaging system's higher acquisition
rate. However, changes in [Ca2+] at low
frequencies of stimulation were less easily detected than with Calcium
Green-1. The procedures for using fura-2 in crayfish axons and for
calibrating it for intraterminal Ca2+
([Ca2+]i) have
been described thoroughly in several previous publications (Delaney et
al., 1989; Delaney and Tank, 1994; Tank et al., 1995; Ravin et al.,
1997). In general, the procedures used in the present study were
similar to those given by Delaney et al. (1989) and Ravin et al.
(1997). In brief, fura-2 pentapotassium salt (Molecular Probes) was
diluted to 20 mM in a solution containing 100 mM KCl and 20 mM HEPES, pH 7.4. The tonic or
phasic axon was penetrated with a single-barrel thick-walled glass
microelectrode (20-40 M when filled with 3 M KCl)
containing the fura-2 dye solution. The neurons were filled
iontophoretically by passing 10-15 nA negative DC current for 10-15
min. The fura-2 was allowed to diffuse fully into the nerve terminals
for 30 min after injection. After this diffusion period, when stable
dye fluorescence was observed in boutons close to the injection site,
nerve terminals were readily imaged within 1-2 mm of the injection
site. Axons were stimulated selectively by varying the voltage applied
through a suction electrode in which they had been placed. The phasic
or tonic axon was stimulated with a brief train, 10 sec in duration, at
2, 5, 10, 20, 30, 40, or 50 Hz (tonic axons received additional trains
at frequencies of 75, 100, and 200 Hz), followed by a 1 min rest period.
[Ca2+] was determined ratiometrically
from a standard imaging system fitted with bandpass excitation filters
of 360 ± 10 and 385 ± 5 nm in a filter wheel, and a
530 ± 35 nm bandpass emission filter (Omega Filters). The
terminals were visualized using an Olympus 40× water immersion
objective, and frames were captured using an intensified CCD camera
(IC-100, PTI) at four frames/sec, using AIW 2.1 (Axon Instruments,
Foster City, CA).
Images were acquired by briefly exposing (33 msec per image) the
preparation to excitation wavelengths of 360 and 385 nm. One image was
acquired at 360 nm (the isobestic point of fura-2) followed by
successive images at 385 nm. The 360 nm image was divided by successive
385 nm images (pixel by pixel), thus creating a series of ratio images.
Background fluorescence was determined from a region of muscle near the
nerve terminals that were imaged and was subtracted from both 360 and
385 nm images before ratios were calculated. As noted previously in
Delaney et al. (1989), autofluorescence of the exoskeleton was
appreciable; therefore terminals were imaged from areas with relatively
low background fluorescence. For determination of resting
[Ca2+], individual terminals were
selected, and the mean ratio for each specified terminal was
determined. Ratios were then averaged over a 5 sec acquisition period.
Resting and stimulus-evoked ratios for each nerve terminal were
converted into an absolute calcium concentration using Equation 5 of
Grynkiewicz et al. (1985).
Fura-2 ratiometric data were calibrated in vitro using a
commercially available kit (Molecular Probes fura-2 Calcium Imaging Calibration Kit). Solutions containing 50 µM
fura-2, 100 mM KCl, and defined
[Ca2+] ranging from 0 to 39 µM were imaged in 50 µm quartz capillary tubes (Vitro Dynamics). Ratios for zero
[Ca2+]
(Rmin) and saturating
[Ca2+]
(Rmax) were determined after
background fluorescence from a fura-2-free solution had been
subtracted. A viscosity correction factor of 0.7 was applied to
Rmax and
Rmin (Delaney et al., 1989; Poenie,
1990). To further verify the results of the in vitro
calibration, an in vivo calibration was also performed,
generally following the procedure found in Ravin et al., (1997).
Rmin was determined in fura-2-loaded
terminals after the preparation was incubated in calcium-free Van
Harreveld's solution with 2 mM EGTA and 10 µM ionomycin for 1 hr. The ratio decreased
slowly and was stable after 1 hr, at which point
Rmin was measured. To establish
Rmax, the bathing solution was changed
to Van Harreveld's solution containing 50 µM
carbonyl cyanide m-chlorophenyl hydrazone to release
[Ca2+] from mitochondria, and 10 µM ionomycin. The ratio increased rapidly and
reached a maximum in <5 min, after which
Rmax was measured. Calibration values
obtained from the in vivo procedure were similar to those
obtained from viscosity-corrected in vitro calibrations. The
values of Rmin and
Rmax were substituted into Equation 5 of Grynkiewicz et al. (1985), along with the value Sf/Sb
(zero calcium fluorescence at 385 nm divided by saturating calcium
fluorescence at 385 nm) and Kd of the
fura-2 in crayfish nerve terminals [865 nM
according to Delaney et al. (1991)].
To reduce twitching of the muscle at high stimulation frequencies, a
glutamate receptor blocker, Jorotoxin (JSTX, Calbiochem, La Jolla, CA),
was applied to the preparation at a concentration of 50 µM diluted in Van Harreveld's solution for 15 min
(Quigley and Mercier, 1997). This effectively blocked
contraction of the muscle, whereas it left presynaptic calcium signals unaltered.
Ultrastructural analysis. For transmission electron
microscopy, samples of axons and neuromuscular junctions were obtained using standard fixation, embedding, and sectioning procedures (Jahromi
and Atwood, 1974; King et al., 1996). Procedures for electron
microscopy of freeze-fracture replicas were similar to those used
previously for other crustacean neuromuscular preparations (Pearce et
al., 1986; Walrond et al., 1993; Govind et al., 1994, 1995).
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RESULTS |
Quantal release from visualized tonic and phasic terminals
Differences in transmitter release were shown by comparing
synaptic currents at small, well defined regions of the nerve terminal. We compared neurotransmitter release at recording sites where phasic
and tonic terminals were close together on the same muscle fiber and
could be assayed with a single placement of the recording electrode
(Msghina et al., 1998). A focal macropatch electrode (15-20 µm) was
placed under visual control over a single prominent tonic bouton and
adjacent phasic terminals (Fig.
2A) to record the
presynaptic nerve terminal spike together with spontaneous and evoked
tonic and phasic quantal events (Fig.
2B,C) from the same patch. The
tonic axon was first selectively stimulated at 1-20 Hz, providing data
on evoked quantal release (Fig.
2B,D). At stimulation frequencies
below 5 Hz, tonic terminals released very few quanta. When quantal
events occurred, they were mostly single events and similar in size to
spontaneously occurring EJCs, which were taken to represent single
quantal events (Fig. 2B). The small numbers of evoked
quantal events made it feasible to count the number of quantal units
evoked by each impulse of the series (Cooper et al., 1995b).
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Figure 2.
Focal recordings of synaptic currents from phasic
and tonic nerve terminals at 1 Hz. A, Recording location
for conjoint assay of phasic and tonic transmission on a single muscle
fiber. Phasic (P) and tonic
(T) nerve terminals were vitally stained with
4-Di-2-Asp and visualized under the confocal microscope. A major
secondary nerve branch (N) containing
the two motor axons crosses the muscle fibers transversely, giving off
smaller tertiary synapse-bearing branches from which recordings were
made. A recording site for the "macropatch" electrode, connected to
the recording preamplifier, is diagrammed. The primary tonic axon
(TA), ~25 µm in diameter, appears in the top
right-hand corner. B, C, Focal
recordings made sequentially from tonic and phasic terminals at a
single site. The tonic and phasic primary axons were stimulated at 1 Hz. Extracellular nerve terminal spikes are indicated by
asterisks. B, Eight superimposed records
of selected responses to stimulation of the tonic axon; two evoked
excitatory junctional currents (EJCs) and one "late
release" or miniature excitatory junctional current
(mEJC) appeared. The majority of stimuli evoked no
response at this frequency. C, Four superimposed records
of selected responses at the same site to stimulation of the phasic
axon. Four late releases (mEJCs) are shown. All stimuli
evoked multi-quantal EJCs. D, Plot of EJCs
(current-time integrals) produced by phasic ( ) and tonic ( )
nerve terminals in response to 1000 stimuli at the site recorded from
in B and C. EJC measurements are plotted
against sweep number. Numerous failures of release occur in the tonic
response; none occur in the phasic response. Gradual depression is
evident over time in the phasic responses and is indicated by a
regression line.
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When the phasic axon was recruited, a markedly different result was
obtained. All impulses of a series released multiple quantal units and
frequently "late events" in the form of individual quantal units
(Fig. 2C). At 1 Hz, phasic EJCs were up to 18 times the amplitude of individual quantal events, indicating a large quantal content at low frequencies of activation (Fig.
2C,D). Generally, transmitter release declined
slowly during maintained stimulation at 1 Hz (Fig.
2D). For comparison, recordings were also made from isolated phasic terminals unaccompanied by a tonic terminal; the general result was the same.
Estimates of quantal content at 1 Hz for paired phasic and tonic
terminals were made in several experiments to compare the effectiveness
of single nerve impulses in releasing quantal units. For the tonic
terminals, all quantal units that were released could be accurately
counted. For the phasic terminals, many quantal units were released per
impulse, and accurate counts of the number of individual quantal events
could not be made. Estimates of mean quantal content for phasic
terminals were derived by dividing the mean evoked response by the mean
quantal unit amplitude. An additional measure of relative output at the
same site was provided by averaging the evoked EJCs and comparing their
amplitude-time integrals (Cooper et al., 1995b; Msghina et al., 1998).
As illustrated in Figures 2D and
3B,D,
these estimates always showed a large difference in quantal release for
phasic and tonic terminals at the same recording sites. Phasic/tonic
ratios of the quantal content at 1 Hz ranged from 94 to 1300 in seven
experiments, with a mean value of 208 (Table
1).
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Figure 3.
Frequency facilitation at tonic (A,
B) and phasic (C, D) nerve
terminals. A, Averaged records of EJCs from a tonic
terminal at four different frequencies (1, 2, 5, 10 Hz). In each case,
500 responses were averaged. Extracellular nerve terminal spikes are
indicated by the asterisks. B, Graph of relative changes
in the averaged EJC (current-time area measurements) over the
frequency range of 1-20 Hz. C, Averaged records of EJCs
from a phasic terminal at three different frequencies (1, 2, 5 Hz; 500 responses for each frequency). D, Graph
of relative changes in the averaged EJC for the phasic terminal ( )
and the tonic terminal () over the range of 1-5 Hz; measurements
were normalized to the values at 1 Hz to make the phasic-tonic
comparison. A marked difference in frequency facilitation is
apparent.
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Short-term facilitation (frequency facilitation) of transmitter release
was also compared for the two terminals using the steady-state EJC
attained during brief trains as a measure of facilitation. During
repetitive stimulation at different frequencies ranging from 1 to 20 Hz, there was a very large facilitation of transmitter release from
tonic terminals (Fig. 3A,B). From 1 to 20 Hz, quantal content increased by up to 100-fold, and many stimuli now caused the release of two to four quanta at 20 Hz. Phasic terminals, on the other hand, showed much less facilitation when tested
at 1-5 Hz (Fig. 3C,D). At frequencies >5 Hz,
the signals at phasic terminals were often contaminated by events at
nearby synapses outside the lumen of the recording electrode, which
resulted in a mixture of positive- and negative-going signals. These
contaminating signals have previously been described and analyzed in
detail at crayfish synapses by Dudel (1965) and Ortiz (1972). In
addition, muscle movement was a significant problem at the higher
frequencies. Therefore, phasic events were not studied in detail at
frequencies higher than 5 Hz. The ratio of transmitter release at 5 and
1 Hz for phasic terminals was always <2 (Fig.
3C,D), whereas for tonic terminals, the ratios
were always >2 (Fig. 3D).
Even with the pronounced frequency facilitation, tonic terminals still
released less transmitter at frequencies of 10 and 20 Hz than did
phasic terminals at 1 Hz. The mean quantal content for phasic terminals
at 1 Hz was 9.5 (Table 1). Tonic terminals facilitated tenfold at 10 Hz
(Fig. 3B). Thus, the value found at 1 Hz (0.055, Table 1)
would rise to a mean quantal content of ~0.5 at 10 Hz. This value is
much lower than that for phasic terminals at 1 Hz. This large
difference in release can be compared with the relative calcium signals
of the two terminals when considering the importance of calcium entry
for determination of synaptic strength in the two axons.
Calcium responses in tonic and phasic terminals
Calcium entry to the active zones of synapses is believed to be
the trigger for evoked release of transmitter (for review, see
Augustine and Charlton, 1986; Llinas et al., 1992; Zucker, 1996;
Stanley, 1997), and one possible reason for the difference in synaptic
strength of phasic and tonic axons is that there is more calcium entry
at synapses of phasic terminals. If there is a third- to fourth-power
relationship between calcium entry at the synapse and immediate
transmitter release, as has been proposed in previous studies (Zucker
and Lara-Estrella, 1983; Augustine and Charlton, 1986; Zucker, 1996), a
200-fold difference in quantal release at 1 Hz (Table 1) would imply a
five- to sixfold greater calcium entry per synapse in phasic terminals.
To test this hypothesis, we estimated the relative calcium entry and
then compared this with the estimated number of synapses and putative
synaptic calcium channels from ultrastructural studies. The combined
physiological and ultrastructural data were used to compare the average
calcium entry per synapse for the two terminals at low frequencies of activation and to assess the likelihood that Ca channels of
phasic terminals are more effective in admitting
Ca2+.
Intraterminal calcium accumulating during impulse activity can be
estimated using calcium indicators injected into the motor axon's
preterminal branches (Delaney et al., 1989; Cooper et al., 1995a). The
calcium so measured can be described by a single-compartment model in
which [Ca2+]i has
reached equilibrium throughout the terminal <100 msec after entry at
synapses, and in which influx and efflux are in equilibrium during a
train of stimuli (Tank et al., 1995). A linear relationship between
[Ca2+]i and
stimulation frequency is an outcome of this model. The single-compartment model provides a good theoretical basis for comparing the calcium signals in the two terminals during short trains
of impulses.
Observations of
[Ca2+]i during
stimulation were initially made with Calcium Green-1, which gives
easily detectable signals at low frequencies (Cooper et al., 1995a).
Subsequent measurements were made with fura-2, which allows
[Ca2+]i to be
estimated ratiometrically. Resting levels of
[Ca2+]i as well as
stimulus-evoked
[Ca2+]i were
estimated with fura-2.
Values for the resting levels of
[Ca2+]i were made
by ratiometric imaging of individual boutons (synapse-bearing
varicosities) and axon branches in several preparations. Phasic and
tonic terminals were easy to distinguish because they differ greatly in
morphology (Fig. 2A) (Bradacs et al.,
1997). The mean values (±SEM) for resting [Ca2+]i were
145 ± 4.5 nM (n = 91, 5 specimens) for tonic boutons and 207 ± 8.5 nM (n = 71, 3 specimens) for
phasic boutons; the means were significantly different statistically
(p 
0.01). These values were used for
estimating changes in calcium during impulse activity from fluorescence
changes of Calcium Green-1.
Two sets of observations of calcium signals during impulse activity
were made on nerve terminals injected with the two different calcium
indicators, Calcium Green-1 and fura-2. For the Calcium Green-1 images,
the motor axons were successively stimulated for 8 sec periods at
progressively higher frequencies over the range of 1-50 Hz. These
short stimulation periods were sufficient for [Ca2+]i to attain
a steady level but not long enough to produce complications caused by
delayed leakage of Ca2+ from mitochondria
after stimulation at low frequencies (Tang and Zucker, 1997).
In tonic terminals, a small calcium signal
(F/F) was detected at stimulation
frequencies below 2 Hz, and as the frequency was raised, the signal
continued to increase up to 50 Hz (Figs. 4, 5). Phasic terminals showed an easily
detectable signal at 1 Hz and at all frequencies tested had a larger
calcium response than tonic terminals (Figs. 4, 5). At the higher
frequencies, some of the records showed irregularities possibly caused
by movement during the imaging (Fig.
5B); therefore, in these
measurements comparisons between phasic and tonic terminals were
limited to the lower frequencies (1-10 Hz).
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Figure 4.
Calcium imaging of phasic and tonic terminals
showing examples for Calcium Green-1 (I, top
panels) and fura-2 (II, bottom
panels). I. Calcium Green-1 imaging, The
preparations were stimulated for 8 sec periods at frequencies ranging
from 1 to 50 Hz. A, Phasic terminals; B,
tonic terminals. In both cases, the first image, labeled 0, represents the control (unstimulated) condition. Frequencies
(Hz) are indicated for each image. Scale bar, 12.5 µm.
II. Fura-2 imaging, Calibrated ratio images are shown.
The preparations were stimulated for 10 sec periods at frequencies
ranging from 2 to 40 Hz, followed by a rest period of 1 min after each
period of stimulation. A, Phasic terminals;
B, tonic terminals. Frequencies (Hz) are indicated for
each image. Phasic terminals show more Ca2+
accumulation at a given frequency. Intraterminal
[Ca2+] can be estimated by using the
calibrated color bar. Scale bar, 10.0 µm.
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Figure 5.
Frequency dependence of the relative calcium
signals (F/F) from phasic and
tonic terminals injected with Calcium Green-1 and estimated changes in
intraterminal [Ca2+] with frequency. Individual
tonic (A) and phasic (B)
terminals were followed in an experiment in which the axons were
stimulated sequentially for 8 sec periods at frequencies ranging from 1 to 50 Hz, as indicated. The fluorescence values were normalized to the
unstimulated (control) response (c) for
each set after subtracting the background fluorescence from both
resting and stimulated fluorescence values. C, Summary
of data from 17 tonic and 23 phasic terminals measured as shown in
A and B; data obtained at 1-20 Hz are
shown. D, Frequency dependence of the calcium signals,
shown by averaging the normalized fluorescence measurements of the
eight frames obtained at each frequency in C. Note that
the relationship is curvilinear. E, Estimated changes in
calcium ([Ca2+]) at frequencies of 1-10 Hz,
obtained from D by applying calibration of Calcium
Green-1 fluorescence (Fig. 1). The increase in
[Ca2+] is approximately linear with frequency.
Average values for quantal content ( ) from Table 1 are indicated
for the tonic terminals at 10 Hz and for the phasic terminals at 1 Hz,
illustrating that quantal output for tonic terminals is 24 times lower,
although the estimated [Ca2+] is four times
higher. F, Relative facilitation values plotted against
calcium signals (F/F) in phasic
and tonic terminals at 1, 2, and 5 Hz. Values normalized to those at 1 Hz. Increased calcium accumulation is accompanied by very little
facilitation in phasic terminals, whereas tonic terminals show a large
increase in facilitation as calcium signals increase with frequency of
stimulation.
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Measurements with fura-2 showed a clear difference in
[Ca2+]i between
phasic and tonic terminals at any given frequency (Figs. 4, 6). In
general, calcium signals at low frequencies were harder to detect with
fura-2 than with Calcium Green-1, but measurements at the higher
frequencies were more accurate because of the higher Kd of fura-2.
As indicated in Figures 4-6, the calcium signal was larger in phasic
terminals at all frequencies for both indicators. When the change in
calcium signal is plotted against facilitation of transmitter release,
a large difference between the two axons is apparent (Fig.
5F). Despite increasing calcium accumulation at
higher frequencies, transmitter release remains close to its 1 Hz value
in phasic axons, implying that the synapses are operating close to
their maximum capacity at 1 Hz. Therefore, factors other than calcium
entry limit transmitter output at phasic terminals. In contrast, tonic
terminals increase their transmitter release with frequency in parallel
with increased total calcium entry and accumulation, but even at 20 Hz
or more they do not approach the quantal release values seen at phasic
terminals. The relative calcium signal is considerably larger in tonic
terminals at 10 and 20 Hz than in phasic terminals at 1 Hz, indicating
that tonic terminals release less transmitter for a given change in
[Ca2+]i (Fig.
5E).
For the Calcium Green-1 data, estimates of the change in calcium with
stimulation (Ca2+) were obtained by
using the resting
[Ca2+]i values
from the fura-2 measurements and applying the semilogarithmic relationship between F/F and
[Ca2+]i (Fig. 1).
For both terminals, a nearly linear relationship between
Ca2+ and frequency resulted (Fig.
5E). This is in accord with the linear relationship between
Ca2+ and frequency reported by Tank et
al. (1995) for the crayfish opener inhibitory axon's varicosities.
The fura-2 measurements also yielded a linear relationship between
Ca2+ and frequency over a wider
frequency range (Fig. 6). The values of
Ca2+ were similar for the two sets of
observations, but because these were obtained from different lots of
crayfish, identical results would not be expected. Results for the
phasic terminals agreed closely, whereas values for tonic terminals
were higher in the Calcium Green-1 estimates. The calcium measurements
represent an average intraterminal change after collapse of the
transient calcium domains near the activated calcium channels, as
discussed extensively by several authors (Delaney and Tank, 1994; Tank
et al., 1995). In our records, a steady-state value of the calcium signal was reached rapidly (Figs. 5, 6). The steady-state level is not
sensitive to the concentration of calcium indicator in the terminal; it
represents the outcome of a balance between influx and efflux (Tank et
al., 1995). In crayfish opener muscle inhibitory axon terminals, the
steady-state calcium change is linearly related to stimulation
frequency (Tank et al., 1995). For the extensor muscle motor neurons,
the relationship was also linear for both phasic and tonic terminals
(Figs. 5, 6). Thus, the single-compartment model of Tank et al. (1995)
is a good initial representation of calcium dynamics in the extensor
motor terminals.
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Figure 6.
Averaged calcium signals from several fura-2
injected phasic (A) and tonic
(B) terminals during brief trains of stimulation
at different frequencies. Stimulation frequency is shown over each
trace; solid bar indicates the period of
stimulation. The relationship between Ca2+ and
frequency (C) is linear for both types of bouton.
Data points were averaged from 5-12 different boutons in three
preparations and show mean ± SEM. The slopes of the two lines
differ approximately fivefold. Over the frequency range of 1-10 Hz,
the values estimated from fura-2 agree well with those estimated from
Calcium Green-1 (Fig. 5).
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Influence of varicosity size on
Ca2+ signals
We investigated the relationship between bouton size and
Ca2+ for the two axons. When the
calcium signal was measured in different-sized boutons of the same
tonic terminals under the same conditions of stimulation, there was no
discernable influence of bouton size (Fig.
7A). In addition, no size
effect was found among varicosities of several different preparations
when F/F or
[Ca2+]i was
plotted against the estimated volume of the varicosity. This is best
shown in a series of fura-2 measurements (Fig. 7B). In
comparing phasic and tonic terminals, it was clear that even when
phasic and tonic varicosities of similar size were selected, the
Ca2+ values were always larger for
phasic varicosities at any given frequency (Fig. 7B). These
observations indicate that within each type of bouton, calcium entry is
scaled to the size of the varicosity. In varicosities, calcium entry
occurs predominantly at synapses (Delaney et al., 1989). In previous
work, it was found that within one type, larger varicosities have more
synapses than smaller varicosities (Quigley et al., 1999); this could
explain the lack of effect of varicosity size on
Ca2+.
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Figure 7.
Analysis of the effect of bouton volume on calcium
signal. A, Example from a Calcium Green-1-injected tonic
terminal in which calcium responses were measured in varicosities of
different sizes (arrows). Scale bar, 12.5 µm. The
graph shows development of calcium signals
(F/F) in the three
different-sized tonic varicosities ( , , ) over the frequency
range of 1-20 Hz, relative to the control (resting value, indicated by
c). The varicosities ranged in size from 2 to 6 µm in
diameter. There were no differences in relative calcium changes in
boutons of different size along the same terminal over the frequency
range of 1-20 Hz. B, Calcium signals in tonic and
phasic varicosities of different volumes from fura-2-injected axons.
The tonic and phasic populations are clearly different, and the calcium
signals do not depend on the size of the bouton within the tonic and
phasic subclasses. Note that tonic and phasic boutons of similar size
have markedly different calcium signals.
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The scaling of calcium entry differs for phasic and tonic boutons,
because even when boutons are of similar size,
Ca2+ is always larger for phasic
boutons at a given stimulation frequency. Neuron-specific scaling of
synaptic spacing on boutons has been documented for
Drosophila (Meinertzhagen et al., 1998) and apparently occurs also in crustacean phasic and tonic terminals, as indicated in
Table 3.
Time course of Ca2+ signals
Different rates of Ca2+ accumulation
could be caused by differences in Ca2+
extrusion (Tank et al., 1995). We therefore compared the rate of
decline of Ca2+ in the two terminals
when peak Ca2+ was the same in both. In
addition, the initial rate of rise of Ca2+ was compared.
When the peak Ca2+ was the same for the
two terminals, the rate of rise of the signal was almost identical
(Fig.
8A,B).
According to the single-compartment model (Tank et al., 1995),
similarity in the initial rate of rise of the signal implies that the
initial buffering of
[Ca2+]i is similar
for the two boutons. Thus, our present measurements do not indicate a
difference in fast buffering, although total buffering capacity may be
greater for the tonic axon, as shown by differential responses to
calcium overload (Atwood and Lnenicka, 1992). The initial rate of rise
of the signals varied with frequency, as expected, and was greater for
the phasic axon at any given frequency, as would be the case
for a larger impulse-linked calcium entry per volume of terminal
(Fig. 8C).
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Figure 8.
Comparison of the kinetics of calcium signals in
phasic and tonic boutons of fura-2-injected axons. A,
B, Two examples of representative records in which peak
calcium was the same for the two boutons; signal rise times are almost
identical, but the decay rate is slower for the phasic axon.
C, Relationship between rate of rise of
[Ca2+]i and frequency for the two
axons; the phasic axon shows a more rapid rate of rise at any given
frequency.
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In contrast, the rate of decline of
[Ca2+]i at the end
of a train was invariably more rapid for the tonic varicosities for a given calcium load (Figs. 8, 9). The estimated time constant for the
initial decay of
[Ca2+]i was
approximately twice as large for the phasic axon (Table 2). This difference in initial rate of
extrusion will influence the comparison of calcium entry for the two
terminals, according to the one-compartment model of (Tank et al.
1995). The more efficient removal of
[Ca2+]i in tonic
axons accords with their normal pattern of maintained impulse
production during locomotory activity, in contrast with phasic axons,
which fire infrequently (Bradacs et al., 1997). A difference in
Ca2+ extrusion has also been seen in
phasic and tonic growth cones (Lnenicka et al., 1998).
There was evidence for two different processes in calcium removal (Fig.
9). At low frequencies,
[Ca2+]i decayed
with a single exponential, but at higher frequencies two decay
processes became evident. The slower phase of decay appeared at much
lower frequencies for the phasic boutons. When the tonic axon was
stimulated at a high frequency to induce a large calcium load, very
similar decay kinetics, with two prominent phases, became apparent. The
slow component may represent reaction of mitochondria (Tang and Zucker,
1997) or another unidentified process. It was not investigated further
in the present study, and only the initial decay rate was taken into
account in comparing bouton calcium entry at low frequencies.
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Figure 9.
Comparison of the rates of decay of
[Ca2+]i in phasic and tonic boutons at
the end of a stimulus train in fura-2-injected axons.
[Ca2+]i is normalized to a percentage
of peak [Ca2+]i. A,
B, Decay of [Ca2+] in boutons
receiving stimulus trains of equal frequency. At frequencies above 5 Hz, decay of [Ca2+]i from phasic
boutons is best described by a double-exponential model, made apparent
by a pronounced slow phase. Conversely, tonic boutons exhibit decay of
[Ca2+]i, which is best
described by a single exponential; however, tonic boutons did display a
pronounced slow decay phase when subjected to a relatively high calcium
load at frequencies above 50 Hz. Representative traces averaged from
several tonic and phasic boutons stimulated at 20 or 40 Hz are shown,
with nonlinear regressions and decay time constants. Note the slow
phase of decay exhibited by phasic terminals at these frequencies.
C, D, Decay of
[Ca2+]i from tonic and phasic boutons
subjected to similar peak calcium loads. Under similar peak calcium
loads, tonic boutons display a more rapid initial decay of
[Ca2+]i. In the two representative
cases, averaged from several boutons, initial decay was approximately
twice as fast for tonic boutons. Data are given for two cases in which
peak [Ca2+]i was similar for both
types of boutons. Table 2 shows this effect over a wider range of peak
[Ca2+]i. Nonlinear regressions are
shown with initial decay time constants.
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It was of interest to know whether the amount of Calcium Green-1 or
fura-2 in the terminals influenced the
Ca2+ removal rate, because the calcium
indicator can affect the kinetics of extrusion (Tank et al., 1995).
Among the varicosities in which the rate of extrusion had been
estimated, several of similar size were found in which the resting
fluorescence values differed. Plotting these values against the
estimated extrusion rates showed no apparent relationship: varicosities
with almost the same extrusion rates had different resting fluorescence
values in several cases. Given that in many cases mean resting
fluorescence values were not greatly different for phasic and tonic
terminals, it is not likely that differences in concentration of the
calcium indicators could have produced the consistently observed
difference in time constant for the decay of
Ca2+ reported in Table 2. Additionally,
fura-2 dye concentration was similar for both axons because
fluorescence excited at 360 nm (which is insensitive to
[Ca2+] but sensitive to dye
concentration) was comparable between them.
Ultrastructure of synapses
To determine the morphological counterparts for the physiological
measurements (quantal content and calcium signals), we estimated the
number of synapses and putative channel particles at synaptic active
zones for the two terminals. In previous studies, we had tested the
hypothesis that differences in the number of synapses or their size
might be correlated with the higher quantal output of transmitter at
the recording sites but found that these features could not account for
transmission differences (King et al., 1996; Msghina et al., 1998). In
fact, the number of synapses per varicosity or per unit length of
terminal is larger for the tonic terminals, and mean synapse size is
larger. Clearly, at 1 Hz the quantal output per synapse is much greater
for phasic terminals. In the present study, we undertook additional
freeze-fracture studies to determine whether it was likely that
individual phasic synapses have more calcium channels in their
active zones than tonic synapses. The occurrence of more calcium
channels, if substantiated, could be one factor accounting for the
difference in calcium signals, which might be linked in turn to
the differences in transmitter release.
A major problem in examination of the freeze-fracture replicas was
identification of phasic and tonic excitatory terminals and also
inhibitory terminals that occur in limited regions of the specimens
(Msghina and Atwood, 1997). Distinctions between excitatory and
inhibitory terminals were made on the basis of previously elucidated
differences in their postsynaptic receptor sites (Franzini-Armstrong,
1976; Pearce et al., 1986; Govind et al., 1995). Receptor site
particles of excitatory axons, both phasic and tonic,
characteristically appear on the muscle external (E)-face aligned in
very regular rows, although occasionally the row-like arrangement is
broken (Fig. 10A).
Inhibitory receptor site particles, on the other hand, are more
prominent on the muscle P-face where they are also regularly
aligned, but in doublet rows (Fig. 10B).
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Figure 10.
Intramembrane features of tonic synapses and
active zones. A, B, Identification of
excitatory and inhibitory synapses in freeze-fracture replicas.
Receptor site particles occur in regular rows on the muscle E-face for
the excitatory axon (A) but as doublet rows on
the muscle P-face for the inhibitory axon (B).
C, Part of a large varicosity of the tonic axon
replicated on the axolemmal P-face, which is rich in particles. This region shows three synapses (delineated by small
arrows), each slightly elevated and containing relatively few
particles except for collections of large particles at putative active
zones (arrowheads). D, P-face view of two
putative active zones (arrowheads) with a circular
cluster of large active zone particles and several small circular
depressions along the periphery representing attachment sites of
synaptic vesicles. E, Thin section of a grazing view of
a presynaptic dense bar (arrowhead) with synaptic
vesicles docked around its periphery. F, E-face of
active zone (arrowheads) with a circle of small
dome-shaped protrusions representing attachment sites of synaptic
vesicles surrounding a particle-free clear area. Scale bars:
A, B, 0.2 µm; C-F, 0.4 µm. Magnification: A, B, 141,000×;
C-F, 49,000×.
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After this initial identification, fracture planes through the
protoplasmic (P)-faces of the presynaptic terminals were particularly helpful in characterizing the synapses and active zones of the two
excitatory axons. Identification of phasic and tonic nerve terminals in
freeze-fracture replicas was based largely on size differences between
them, especially when they occurred close to each other (Bradacs et
al., 1997). As seen in Figure 2A, and in accordance
with previous studies, a consistent difference in overall morphology
was apparent: phasic terminals are more slender and lack the prominent,
relatively large varicosities of the tonic terminals (Atwood and
Cooper, 1995; King et al., 1996; Bradacs et al., 1997). In transmission
electron micrographs, synaptic contacts on both are qualitatively
similar, as are active zones comprising presynaptic dense bars around
which synaptic vesicles are docked (Fig. 10E). The
putative calcium and calcium-activated potassium channels at these
active zones become visible in the surface morphology of the synaptic
membrane when it has been fractured along its lipid bilayer (Heuser and
Reese, 1973; Heuser et al., 1974; Rheuben and Reese, 1978; Pearce et
al., 1986; Walrond et al., 1993). However, to be certain of the
identity of the terminals, we used only those that could be clearly
identified as excitatory and for which the overall phasic-tonic
differences in morphology were clearly apparent; these constraints
severely limited the number of acceptable specimens.
The tonic terminals appeared as relatively large varicosities (diameter
2.20 ± 0.97 µm, n = 8, mean ± SD in the
present sample) with several synapses (Fig. 10C). Synapses
were defined by roughly circular or oval elevations (plaques) that,
except at the active zones, were relatively particle-sparse compared
with the neighboring axolemma. Clusters of large particles indicating
locations of ion channel structures (thought to be primarily calcium
channels together with some calcium-activated potassium channels)
denoted active zones, as in other well studied synapses (Pumplin et
al., 1981; Roberts et al., 1990). Around the periphery of these active zones were occasional small circular depressions indicative of vesicle
attachment or fusion sites (Fig. 10D). These images
of fusion are induced and accumulated during chemical fixations and do
not indicate the normal quantal release of the synapse but rather the
preferred sites of vesicle fusion. The array of fusion images
corresponds to the circle of docked vesicles around the presynaptic
dense bar seen in thin sections (Fig. 10E). The same organization of the active zone was seen on the E-face of the fractured
presynaptic membrane where vesicle attachment sites often appeared in a
circle around a slight depression representing the active zone (Fig.
10F). These tonic active zones were circular in shape
and contained on average of ~15 large membrane-associated particles
(range 12-17; n = 5). In the tonic varicosity of
Figure 10C, the larger synapses supported several active
zones, whereas the smaller ones supported a single active zone. In
synapses with multiple active zones, the center-to-center spacing
between adjacent active zones was 217 ± 25 nm (n = 9, mean ± SD).
The thin filiform terminals of the phasic axon (diameter 0.62 ± 0.12 µm, n = 6, mean ± SD in this sample)
showed a serial rather than grouped arrangement of the individual
synaptic contacts (Fig.
11A). Otherwise they
were qualitatively similar in appearance to the tonic synapses. Each
phasic synapse possessed at least one active zone in which the large
membrane-associated particles were clustered in a circular (Fig.
11B,C) or elongated (Fig.
11D) region. In one unusual example, four circular
active zones occurred very close together, creating a long complex
structure (Fig. 11E). In the circular active zones,
there were ~15 large particles (n = 3) but twice this
number in a more distinctly elongated active zone. The center-to-center
spacing between paired circular active zones of the same synapse was
88 ± 46 nm (n = 8, mean ± SD), and this was
significantly smaller (Student's t test, p < 0.05) than the spacing for the tonic synapses.
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Figure 11.
Intramembrane features of phasic synapses and
active zones. A, Fracture through a thin filiform nerve
terminal characteristic of the phasic axon. For most of its 8 µm
length the fracture plane is through the axolemmal E-face
(EF) but then passes through a cross-fracture
(CF) before exposing the axolemmal P-face
(PF). Three synaptic contacts (delineated by small arrows) are seen in
register along the terminal; two are in the E-face and one is in the
P-face. Active zones (arrowheads) occur within each
synapse. B, Synaptic contact with two clusters of large
particles at two closely adjacent active zones (arrows).
From the P-face, the fracture plane jumps to the muscle E-face and
shows the characteristic arrangement of excitatory synaptic receptor
particles (double arrow). C, Synapse with
two active zones (arrows) on a synapse adjacent to which
characteristic receptor zone particles (double arrow)
are seen on the muscle E-face. D, An elongated active
zone (arrow) with a clustering of large particles and a
vesicle fusion image at its edge. E, Enlarged view of a
phasic synapse showing a synaptic contact with a collection of large
particles in a complex structure consisting of four adjacent circular
active zones (arrows). Scale bars: A, 1 µm; B-E, 0.2 µm. Magnification: A,
25,600×; B, 93,000× C, 50,400×
D, 69,750×; E, 105.000×.
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We were able to supplement these observations with additional images
from the fast abdominal flexor muscles, in which tonic innervation does
not occur (Kennedy and Takeda, 1965a,b), making the identification of
phasic terminals much easier. In this material, circular active zones
contained on average 13 (n = 10) particles and often
occurred in closely associated pairs with a center-to-center spacing of
74 nm (n = 12). These observations are in agreement with those for the phasic axon terminals of the leg extensor, which
were more difficult to identify reliably.
Comparison of the collective results for phasic and tonic synapses
indicates no major differences in active zone structure at this level
of observation, apart from the occurrences of a few longer active zones
in the phasic terminals. Also, as observed in transmission electron
microscopy (King et al., 1996), active zones of phasic synapses were
more likely to occur in closely spaced pairs, and in one case four
closely spaced active zones were found. Generally, the results from the
freeze-fracture replicas were in agreement with those from transmission
electron microscopy (King et al., 1996).
We examined the relationship between the number of active zones and the
size of the varicosity in a series of eight (tonic axon) varicosities
reconstructed from serial electron micrographs in this study and
previous ones (King et al., 1996; Msghina et al., 1998). We calculated
the number of active zones per unit volume of varicosity. On average,
there were 1.21 ± 0.26 (SE) active zones per cubic micrometer,
and there was not a significant regression of these values on planar
area or volume of the varicosity. From the data available in this
sample, the ratio of active zones to volume of the varicosity did not
change significantly with size of the varicosity. This accords with the
data on Ca2+ and bouton size (Fig.
7).
The present study provides the first freeze-fracture views of parallel
phasic and tonic crustacean synapses and an opportunity to compare
upper limits of putative channel structures for the two terminals. Many
of the structural features are very similar to those reported
previously for insect synapses (Rheuben and Reese, 1978; Rheuben and
Kammer, 1983; Rheuben, 1985). In both crustaceans and insects, limited
numbers of active zone membrane particles occur in a cluster within the
synaptic plaque, and release of vesicles occurs at edges of the
cluster. In comparing fast (phasic) and slow (tonic) active zones in
the moth Manduca, Rheuben (1985) found that there are more
synapses (and active zones) in phasic junctions than in tonic and that
the number of membrane particles per active zone is almost twice as
great. Both features readily correlate with more vesicle release at
phasic junctions. However, data on relative calcium entry and
transmitter release per synapse are not available for these insect
neuromuscular junctions, and in addition, some differences between them
may relate to their occurrence on different target muscles and to
differences in developmental stage. The differences reported for moth
neuromuscular junctions do not hold for crustacean terminals if the
comparison is made for equivalent lengths of terminal. Tonic terminals
have more individual synapses and active zones per unit length (King et al., 1996) (Table 3), and although
elongated or closely spaced active zones are more common in phasic
terminals, an overall difference in the number of membrane particles
per active zone was not demonstrated in the present study. The number
and arrangement of prominent membrane-associated particles were
approximately the same for similar-sized active zones of the two types
of synapse. The prominent particles are thought to represent synaptic
calcium channels and, in some cases, calcium-activated potassium
channels (Pumplin et al., 1981; Roberts et al., 1990; Pawson et al.,
1998).
In vertebrate (lizard) phasic and tonic motor nerve terminals, active
zone numbers per terminal are similar, but the arrangement of active
zone particles (putative calcium channels) differs greatly and could
influence evoked release [Walrond and Reese (1985); for review, see
Atwood and Lnenicka (1986)]. A comparable difference in active zone
particle placement was not apparent in crayfish phasic and tonic terminals.
Ultrastructural features and calcium entry
An overall comparison of ultrastructural features and calcium
entry for phasic and tonic boutons is presented in Table 3. The number
of active zones per volume of bouton will influence Ca2+, assuming that the calcium
channels per active zone are on average similar for the two types of
bouton. From previ
TOP
ABSTRACT
INTRODUCTION
