Department of Biology, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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Vyshedskiy, Andrey and Jen-Wei Lin. Presynaptic Ca2+ Influx at the Inhibitor of the Crayfish Neuromuscular Junction: A Photometric Study at a High Time Resolution. J. Neurophysiol. 83: 552-562, 2000. Presynaptic calcium influx at the inhibitor of the crayfish neuromuscular junction was investigated by measuring fluorescence transients generated by calcium-sensitive dyes. This approach allowed us to correlate presynaptic calcium influx with transmitter release at a high time resolution. Systematic testing of the calcium indicators showed that only low-affinity dyes, with affinities in the range of micromolar, should be used to avoid saturation of dye binding and interference with transmitter release. Presynaptic calcium influx was regulated by slowly increasing the duration of the action potential through progressive block of potassium channels. The amplitude of the calcium transient, measured from a cluster of varicosities, was linearly related to the duration of the action potential with a slope of 1.2. Gradual changes in potassium channel block allowed us to estimate the calcium cooperativity of transmitter release over a 10-fold range in presynaptic calcium influx. Calcium cooperativity measured here exhibited one component with an average value of 3.1. Inspection of simultaneously recorded presynaptic calcium transients and inhibitory postsynaptic currents (IPSCs) showed that prolonged action potentials were associated with a slow rising phase of presynaptic calcium transients, which were matched by a slow rate of rise of IPSCs. The close correlation suggests that fluorescence transients provide information on the rate of calcium influx. Because there is an anatomic mismatch between the presynaptic calcium transient, measured from a cluster of varicosities, and IPSC, measured with two-electrode voltage clamp, macropatch recording was used to monitor inhibitory postsynaptic responses from the same cluster of varicosities from which the calcium transient was measured. Inhibitory postsynaptic responses recorded with the macropatch method exhibited a faster rising phase than that recorded with two-electrode voltage clamp. This difference could be attributed to slight asynchrony of transmitter release due to action potential conduction along fine branches. In conclusion, this report shows that fluorescence transients generated by calcium-sensitive dyes can provide insights to the properties of presynaptic calcium influx, and its correlation with transmitter release, at a high time resolution.
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INTRODUCTION |
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The crayfish neuromuscular junction was one of the
model synapses in which quantitative calcium imaging was first
implemented to investigate the role of presynaptic free
Ca2+ during posttetanic potentiation (PTP) and
augmentation (Delaney et al. 1989; Delaney and
Tank 1994
). These studies made the important finding that PTP
and augmentation could not be quantitatively accounted for by the
classical residual calcium hypothesis. Measuring the presynaptic
Ca2+ level during serotonin (5-HT)-mediated
synaptic enhancement revealed that 5-HT enhances transmitter output by
mechanisms downstream of presynaptic Ca2+ influx
(Delaney et al. 1991
). The Ca2+
imaging approach has also been used to correlate the relative strength
of Ca2+ influx and the magnitude of synaptic
facilitation (Cooper et al. 1995
). Although these
studies provided unprecedented insights toward our understanding of
Ca2+ dynamics in the presynaptic terminal, the
need to use two excitation wavelengths and/or to process an image
limited the time resolution to the range of seconds. The use of
fluorescence transients generated by calcium-sensitive dyes provides an
opportunity to investigate the Ca2+ transient
with a high time resolution and to infer the kinetics of presynaptic
Ca2+ influx. For example, this approach has
revealed that the delay between Ca2+ influx and
the postsynaptic response is 60 µs at 37°C in the cerebellum
(Sabatini and Regehr 1996
). Furthermore, this technique has been used to demonstrate that synaptic facilitation is mediated by
a Ca2+-driven process that persists after the
presynaptic Ca2+ level has decayed to a low level
(Atluri and Regehr 1996
). Finally, fluorescence
transients of Ca2+ indicators have also been used
to uncover the Ca2+ cooperativity of transmitter
release and the role of different types of Ca2+
channels on the secretion process (Mintz et al. 1995
;
Wu and Saggau 1994a
,b
, 1995
).
Electrophysiological analysis of the presynaptic
Ca2+ current
(ICa) in the crayfish has only been
accomplished recently (Wright et al. 1996a,b
). The
presynaptic ICa exhibits an
intermediate activation threshold and significant calcium-dependent
inactivation (Hong and Lnenicka 1995
; Wright et
al. 1996b
). Pharmacological analysis suggests that transmitter
release and presynaptic ICa can be
completely blocked by the P-type Ca2+ channel
blocker,
-Aga-IVA (Wright et al. 1996b
; see also
Araque et al. 1994
). Correlation between
ICa and excitatory postsynaptic potential (EPSP) has provided an estimated Ca2+
cooperativity of 3 (Wright et al. 1996a
), which is
consistent with a fura-2 imaging study that correlated presynaptic
Ca2+ concentration with the frequency of
asynchronized quantal events (Ravin et al. 1997
). A
similar value for the cooperativity has been estimated indirectly from
decaged Ca2+ in the excitor of the crayfish
neuromuscular junction (Lando and Zucker 1994
). Although
electrophysiological measurement of ICa provides a direct assessment of
calcium influx, implementation of voltage clamp at the crayfish
neuromuscular junction has been difficult. The use of calcium-sensitive
dyes represents a promising alternative for monitoring presynaptic
calcium influx with a high time resolution. In this report,
fluorescence transients generated by calcium-sensitive dyes are used to
monitor presynaptic calcium influx. The time course of calcium
transients is shown to be consistent with known kinetic properties of
presynaptic calcium current. In addition, we show that the presynaptic
calcium transients provide an accurate estimate of the calcium
cooperativity of transmitter release.
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METHODS |
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Preparation and electrophysiology
Crayfish, Procambarus clarkii, were obtained from
Carolina Biological, Burlington, NC. Animals were maintained at room
temperature, 23°C, until use. All experiments were performed at the
same temperature. The typical size of the animals was ~6 cm, head to
tail. The opener muscle of the first walking leg was used for all
experiments. A presynaptic electrode penetrated the major branch point
of the inhibitory axon (inhibitor) to record action potentials and
inject dyes. The major branch point was ~100-300 µm from the
terminals, on a central muscle, where fluorescence transients were
measured. A suction electrode was used to stimulate the inhibitor. Two
postsynaptic electrodes, 5 M with 3 M KCl, penetrated a muscle
fiber. A two-electrode voltage-clamp amplifier (GeneClamp 500, Axon
Instrument) was used to record inhibitory postsynaptic current (IPSC).
We monitored chloride equilibrium potential
(ECl) during experiments to control for possible changes in IPSC due to drifting
ECl (Vyshedskiy and Lin
1997b
).
Control saline contained (in mM) 195 NaCl, 5.4 KCl, 13.5 CaCl2, 2.6 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), titrated to pH 7.4 by NaOH. When tetraethylamonium chloride (TEACl) was introduced into the control saline, an equal amount of NaCl was removed. All chemicals were purchased from Sigma.
Morphological examination of the terminal branches that innervated the
recorded muscle fiber was performed after each experiment, by sketching
or photography (Vyshedskiy and Lin 1997b).
Macropatch recordings were obtained using a patch electrode, containing
the bath solution, with a tip diameter of 20 µm. To place the
macropatch pipette under a ×60 microscope objective, the electrode had
a long shank and approached varicosities with a shallow angle,
~35°. The pipette tip was polished at 35° by beveling (EG-6,
Narishige) such that the rim of the pipette opening could make close
contact with the surface of a muscle fiber. To prevent accumulation of
small particles inside the tip during beveling, which could result in
light scattering, distilled water was continuously forced through the
pipette opening during beveling. Beveling continued until the tip
opening reached ~20 µm. Suction was then applied to the back of the
pipette for 10 min to remove water from the pipette. The pipette was
fire polished before use. In addition, a sharp electrode containing 3 M
KCl, 5 M, impaled the muscle fiber on which the patch electrode made
recordings. The sharp electrode was needed to inject current pulse and
thereby to increase the driving force for chloride. The duration of the current injection was typically 1 s. DC current injection does not
change ECl because crayfish muscle
fibers can adjust ECl to a new
membrane potential in 30-60 s (Finger and Stettmeier
1981
). The patch electrode was connected to a current-clamp
amplifier, Warner IE-210, and inhibitory postsynaptic responses were
recorded as a small change in potential.
Photometric measurements of calcium transients
The inhibitory axon was penetrated at the major branch point by
a sharp electrode containing 5 mM of membrane-impermeable calcium-sensitive dye, potassium salt, dissolved in 400 mM KCl and 20 mM K HEPES (pH 7.4), with a final resistance of 20 M. Five dyes were
tested: Calcium Green-1, Calcium Green-2, Calcium Green-5N, Magnesium
Green, and Oregon Green 488 BAPTA-5N (Molecular Probes). The dyes were
injected by hyperpolarizing current, 2-8 nA, until varicosities close
to the injection site were clearly visible, 10-20 min. It is not
possible to accurately estimate the concentration of injected dyes.
However, we established that the injection protocol did not interfere
with intrinsic calcium buffering or transmitter release (see
below). All dyes could be injected easily except Calcium
Green-2, which invariably clogged the electrode. Calcium Green-5N
behaved erratically, as reported by others as well (Helmchen et
al. 1997
) (and see Fig. 2D) and was not used.
Calcium transients were recorded on an upright microscope (Zeiss
Axioskop) with a ×60 water immersion lens. A tungsten lamp, 12 V 150 W, was powered by a stabilized power supply (Kepco, ATE 15-15DM).
Illumination was gated by a shutter (Uniblitz, Vinsent Associates),
with a typical duration of 600 ms and repeated at 0.2 Hz. This duration
and rate of illumination did not cause significant dye bleaching.
Specifically, there was no detectable decay in the background
fluorescence level during each episode of 600-ms exposure. In addition,
in 10 preparations where the recording lasted from 1.5 to 3 h,
changes in the background fluorescence levels measured at the beginning
(Fb) and the end
(Fe) of the recording sessions were not
statistically different from each other, i.e.,
[(Fb Fe)/Fb] * 100% = 2.68 ± 7.73%. The specifications of the filter set were
485DF22 excitation, 505DRLPO2 dichroic, and 530DF30 emission (Omega
Optical). The area of illumination was defined by an iris diaphragm
custom-milled to allow a smaller diameter opening. Emission was
measured by a photomultiplier tube (PMT; HC124-06, Hamamatsu). The
output of the PMT was filtered with an 8-pole Bessel filter (902LPP,
Frequency Devices) fc = 1 kHz and
digitized at 10 kHz. The cutoff frequency was chosen empirically, based
on the finding that calcium transients measured with
fc of 1 and 4 kHz did not exhibit a
detectable difference in their rising phase. (see also Sabatini
and Regehr 1998
). Fluorescence transients are presented as
F/F = [F(t)
Frest]/Frest *
100%, where Frest represents the background
fluorescence intensity. Experiments commenced after the fluorescence
level had stabilized, ~30 min after finishing dye injection. The
illuminated area was restricted to a ~20- to 50-µm-diam area that
contained one to five varicosities on the central muscle fibers.
Preference was always given to well-isolated and small to intermediate
size varicosities. Only varicosities on the upper surface of the muscle
were investigated. Special care was taken to avoid 3° branches on the
lower surface of the muscle fiber. When a macropatch was used, the
illuminated area was restricted to the varicosities enclosed in the
opening of the pipette. The fluorescence signals thus recorded were not significantly attenuated as long as the pipette tip was free of particles.
Figure 1 illustrates fluorescence
transients recorded from four different areas. The axon was loaded with
Magnesium Green. Calcium influx was evoked by a burst of 20 action
potentials at 100 Hz. When light was concentrated on the area
containing five small varicosities (area 1), Magnesium Green reported a
fast rise and decline in the fluorescence signal. When light was
concentrated on a branch point where a 2° branch gave rise to three
thin branches (area 2), the fluorescence transient showed a slower
decay (· · ·). The fluorescence transient recorded from a
primary branch (area 3) was very small in amplitude and very slow in
time course. When light was concentrated on an area without axon or
varicosities (area 4), stimulation of the inhibitor elicited no change
in the fluorescence transient. These observations are similar to
studies obtained with fura-2 imaging (Delaney et al.
1991) and suggest that fluorescence transients recorded from
varicosities are minimally contaminated by stray signals from nearby
structures.
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We examined the effects of calcium-sensitive dyes on the dynamics of presynaptic calcium transients. The main criteria in selecting a dye included 1) that the dye did not interfere with intrinsic calcium buffering, 2) that the dye did not interfere with transmitter release, and 3) that the dye reported the presynaptic calcium influx linearly.
Interference with intrinsic buffering by calcium-sensitive dyes
can be easily identified as a lengthening of the decay time constant of
the fluorescence transient (Tank et al. 1995). Only high-affinity dyes, such as Calcium Green-1, exhibited a
concentration-dependent effect on the decay time constant of the
calcium transient. For example, the decay time constant of the Calcium
Green-1 transient, activated by 20 action potentials at 100 Hz,
increased from 748 to 1,655 ms when the dye injection period was
increased from 1 to 22 min. This observation suggests that overloading
of Calcium Green-1 interferes with endogenous buffer (Tank et
al. 1995
). Similar results were observed in four additional
preparations. In contrast, decay of the Ca2+ transient
monitored with a low-affinity dye, such as Magnesium Green, remained
unchanged when similar injection durations were compared. Thus the
injection protocol used here for low-affinity dyes, 2-8 nA for 10-20
min, did not interfere with the intrinsic buffering capacity of the
inhibitor terminals. On average, 20 action potentials firing at 100 Hz
were able to activate a 3.2 ± 1.43% (mean ± SD,
n = 22) increase in Magnesium Green-mediated fluorescence signal. The decay time course measured with this dye was
best fitted by a function with two exponential components, with time
constants of 84 ± 30 and 390 ± 153 ms, respectively (n = 22). (In this report, n
represents the number of preparations and averaged data are presented
as means ± SD.)
A large injection of Calcium Green-1, up to 30 min at 6 nA, resulted in a decrease in transmitter release (n = 4). Comparable injection of Magnesium Green (n = 4) or Oregon Green-488 BAPTA-5N (n = 1) did not suppress transmitter release.
The saturation properties of dyes were tested by stimulating the axon
at 100 Hz in control saline. Because the shape of an action potential
does not change up to 100 Hz in control saline, we assume that calcium
influx remains constant for each action potential. Figure
2A, left
panel, illustrates fluorescence transients, generated by
Calcium Green-1, activated by 20, 40, 60, 80, and 100 action potentials
in control saline. The Ca2+ transients produced by each
successive burst of 20 action potentials are shown in the right
panel. The progressive decrease in amplitude of calcium
transients suggests a saturation of Calcium Green-1. However, such a
decrease does not occur with low-affinity dyes such as Magnesium Green
(Fig. 2B) or Oregon Green 488 BAPTA-5N (data not shown).
Extrapolation based on a previous study suggests that the steady-state
[Ca2+]i activated by action potentials at 100 Hz should be ~3.8 µM (Tank et al. 1995), which is
consistent with the fact that Calcium Green-1
(Kd = 190 nM), but not Magnesium Green
(Kd = 6 µM) or Oregon Green 488 BAPTA-5N (Kd = 20 µM), become
saturated. Although one can partially avoid the saturation of
high-affinity dyes by increasing the injected dye concentration, one
then runs into problems of interference with transmitter release.
Therefore low-affinity calcium-sensitive dyes are preferred for
investigation of the effects of large calcium influx. The degree of dye
saturation was consistently related to its calcium affinity. Figure
2D summarizes measurements, based on the protocol used
in Fig. 2, A and B, obtained from the 5 different dyes (see the figure legend for sample size). Here,
successive bursts of 20 action potentials reported by Calcium Green-1
(
) and
2 (
) exhibit a decline in their amplitudes. However,
low-affinity dyes, Magnesium Green (
) and Oregon Green 488 BAPTA-5N
(
), do not show such decline. The saturating behavior of Calcium
Green-5N (
), which is also a low-affinity dye, is unexpected. The
reason for the apparent saturation is not pursued here. All data
presented in the rest of this report were obtained with Magnesium
Green.
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The degree of dye saturation was also examined in the presence of a
high level of potassium channel block, with 1 mM 4-aminopyridine (4-AP)
and 20 mM TEA in saline. Simultaneous application of both potassium
channel blockers increased the duration of action potentials from 0.8 ms to 6-8 ms. First, we compared the calcium transients activated by
single broad action potentials with those activated by 100 and 200 Hz
stimulation in control saline. Figure 2C, left panel,
shows that 1 s of 200-Hz stimulation increases the fluorescence change to nearly 8% (· · ·), which is larger than that
activated by a broad action potential () or by 100-Hz stimulation
(
). The right panel of Fig. 2C shows
that the calcium transients activated by consecutive bursts of 40 action potentials at 200 Hz remain constant and thus show no sign of
saturation. Similar results were obtained from six preparations, and
the averaged data are shown in Fig. 2D (
). Comparison
among the fluorescence transients, recorded from the same varicosities,
activated by 100- and 200-Hz stimulation for 1 s and that
activated by individual broad action potentials was performed in six
preparations. The averaged fluorescence changes activated by 100- and
200-Hz stimulation were 6.2 ± 1.8% and 12.7 ± 4.0%,
respectively. The two values bracket the fluorescence changes activated
by single broad action potentials recorded from the same preparations,
8.0 ± 1.9%. Therefore, if the 200-Hz stimulation protocol showed
no sign of saturating Magnesium Green, calcium influx activated by
individual broad action potentials should not saturate the dye either.
Finally, we sought to activate a maximal change in Magnesium Green
fluorescence intensity in a different series of experiments. One second
of 100-Hz stimulation of the inhibitor in 1 mM 4-AP and 20 mM TEA was
able to elevate the peak fluorescence signal to 21.9 ± 7.7%
(n = 8). In addition, continuous depolarization of
the inhibitor, to 0 mV for 10 s by using the presynaptic voltage
control method (Vyshedskiy and Lin 1997b
), was able to
increase the maximal fluorescence level to 77% (data not shown). Both
sets of data suggest that the typical calcium transient activated by a
single broad action potential is far from the maximal or saturating level.
In this report we try to correlate calcium transients recorded from a cluster of varicosities with IPSCs recorded from the same muscle fiber on which the varicosities were located. This configuration raises the question of a potential mismatch between the two sets of recordings, because the calcium transient is sampled from a subset of varicosities that contribute to the IPSC. We therefore examined the waveforms of calcium transients recorded from different clusters of varicosities (Fig. 3). Clusters identified as areas 1 and 2 represent varicosities originating from separate secondary branches (Fig. 3A, inset) but are located on the same muscle fiber, outlined by the dotted lines. Fluorescence measurements obtained from these two areas are superimposible, during both their rising (B) and decaying (A) phases. The varicosity identified as area 3 has an unusually large diameter, 15 µm. The fluorescence transient recorded from area 3 exhibits a rising phase superimposible with those of areas 1 and 2 (Fig. 3B) but with a slower decay (Fig. 3A). Similar results have been observed in four additional preparations. Because questions addressed in this report do not concern the decay in calcium concentration, causes underlying the difference in the decay time constants are not pursued. More importantly, results shown in Fig. 3 provide sufficient assurance that there is no significant variation, among clusters of varicosities, in the rising phase of calcium transients. Therefore monitoring calcium transients from a small cluster of varicosities provides an adequate representation of presynaptic calcium transients.
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RESULTS |
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Correlation between presynaptic action potential and calcium transients
To analyze the relationship between presynaptic calcium influx and
the duration of an action potential, a broad range of action potential
durations was analyzed by introducing 4-AP and TEA gradually over a
period of 1-2 h. A typical control action potential has a peak level
of +10 mV and a duration of 0.8 ms, measured at 30 mV (Fig.
4B1,
). Addition of 1 mM
4-AP lengthens the duration of the action potential by 0.3 ms and
increases the amplitude slightly (Fig. 4B1, · · ·).
TEA was then introduced, in the continuous presence of 1 mM 4-AP, in
the order 1, 2, 4, 10, and 20 mM. In the presence of 1 mM 4-AP, TEA at
1 mM further increases the duration and creates a persistent
afterdepolarization (Fig. 4B1, - - -). TEA over the
concentration range of 2-4 mM increases the duration of the action
potential further and causes a second spike, Fig. 4B2. (Due
to variations in the delay of the 2nd spikes, their amplitudes appear
decreased, and duration prolonged, as a result of signal averaging.)
The repetitive firing disappears with further increases in TEA
concentration, and action potentials now exhibit a prolonged shoulder
(Fig. 4B3).
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The effects of a progressive increase in K channel blocker concentration are summarized in Fig. 5A. On average, 1 mM 4-AP increases action potential duration by 65%. The introduction of TEA at 1 and 2 mM further increases the duration to 200 and 280% of control level, respectively. There is a drastic increase in action potential duration, to 700%, after the introduction of 4 mM TEA. (The data point marked with * was measured during a transition period, i.e., before the effect of 4 mM TEA had reached a steady state.) The highest concentration of TEA, 20 mM, further increases the duration of the action potential, to 880% of control level. TEA concentrations higher than 20 mM did not lengthen the action potential further but frequently caused instability in membrane potential (data not shown).
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Calcium transients measured simultaneously with the action
potentials just described are shown as the top trace in Fig.
4, A1-A3. The amplitude of the calcium transients increases
with the broadening of the action potentials. Spikes associated with repetitive firing are consistently correlated with additional steps on
the calcium transients (Fig. 4A2). There is a linear correlation, with a slope of 1, between the duration of the action potential, measured at 30 mV, and the amplitude of the presynaptic Ca2+ transient (Fig. 5B,
). Results
obtained from an additional six preparations are also shown in
different symbols, with an average slope of 1.22 ± 0.35 (n = 7). The average amplitude of the calcium transients activated by a single action potential in control saline is
0.43 ± 0.10% (n = 10). Although the quantitative
relationship between action potential duration and
Ca2+ influx appears to be simple, inspection of
individual traces reveals additional complexity. When the duration of
action potential is short, the maximal level of
Ca2+ transient occurs when the action potential
has nearly repolarized to the level of resting membrane potential (Fig.
4, A1 and B1, vertical line). However, with very
long action potentials, the maximal Ca2+
transient occurs when the action potential has repolarized to about
30 mV (Fig. 4, A3 and B3, vertical lines). The
cause of this discrepancy will be discussed later (see
DISCUSSION).
Correlation between the presynaptic Ca2+ transient and the postsynaptic response
Calcium cooperativity was estimated by simultaneously recording
IPSCs and the presynaptic Ca2+ transient. The
magnitude of the presynaptic Ca2+ influx was
changed systematically by slowly introducing K+
channel blockers. Figure 6 illustrates an
example of this type of experiment. An action potential activates a
Ca2+ transient of 0.8% in control saline (Fig.
6, B1 and C1, ). Increases in action potential
duration are accompanied by increases in the amplitude of the
Ca2+ transient (Fig. 6B1,
· · · and - - -) and IPSC (Fig. 6A1, · · ·
and - - -). Presynaptic Ca2+ transients and
IPSCs recorded under extensive block of presynaptic K+ conductance are shown in Fig. 6, A2, A3,
B2, and B3. A consistent finding is that the peaks of
the Ca2+ transients coincide with the peaks of
IPSC. Assuming that the peak of IPSC signifies with the termination of
the release process and calcium influx, the maximal points of
Ca2+ transients then faithfully reflect the end
of Ca2+ influx.
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Data measured from Fig. 6 show that the relationship between the
amplitudes of IPSC and presynaptic Ca2+
transients is nonlinear. A double logarithm plot of the two variables measured from traces shown in Fig. 6 is best fitted by a straight line
with a slope of 2.7 (Fig. 7, and
· · ·). Data obtained from nine additional preparations are
also plotted as different symbols. Most of the data points fall within
the region bordered by lines corresponding to n = 2 and
n = 4 (
). Cooperativity averaged from 10 preparations
is 3.08 ± 0.70 (n = 10; Fig. 7,
and
, experiments where postsynaptic measurements were IPSPs, rather than
IPSCs).
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Additional insights to the close relationship between presynaptic
Ca2+ transients and IPSC are better appreciated
by comparing the rising phase of IPSC and the corresponding
Ca2+ transient in the traces shown in Fig. 6,
A3 and B3. Here the broadest action potential
(Fig. 6C3, - - -) activates a Ca2+
transient (Fig. 6B3, - - -) that exhibits a slower rising
phase than that activated by the next broadest action potential (Fig. 6, B3 and C3, · · ·). The difference
between the Ca2+ transients is presumably due to
a smaller Ca2+ driving force during the plateau
phase of the broadest action potential. This difference is matched by a
similar difference in the rising phase of IPSCs (Fig. 6A3,
- - - and · · ·). [It should be noted that the rising phase
of Ca2+ transients is not limited by dye binding
rate because Magnesium Green has an on-rate time constant in the range
of 100-500 µs (Sabatini and Regehr 1998).]
To further examine how well calcium transients reflect the time course
of calcium influx, we tried to quantitatively correlate the rising
phase of calcium transients with their corresponding IPSCs.
Specifically, we aimed to determine whether we could detect differences
in the rising phase of fluorescence transients when a change in calcium
influx had occurred, as indicated by a shift in the rising phase of
IPSC. Traces in Fig. 8A were
obtained from a preparation different from that used in Fig. 6. Here
the maximal amplitudes of calcium transients and IPSCs have been
normalized to the same height. The calcium transient and its
corresponding IPSC activated by the broadest action potential exhibit
the slowest rising phases (black traces). The difference in time
between adjacent IPSCs at the half-maximal points can be clearly
identified and measured (1-4 in top traces of Fig.
8A). However, due to noise in the fluorescence measurement,
it is not possible to confidently measure the difference between the
half-maximal points of the transients activated by narrow action
potentials (Fig. 8A, bottom traces between yellow and red
traces). A clear correlation can be seen when the difference in
half-maximal points between adjacent IPSCs
(IPSC50) is plotted against the corresponding
difference for calcium transients (
Ca50; Fig.
8B,
with enclosed numbers). The data point corresponding
to the difference identified as 1 in Fig. 8A falls inside an
elongated circle (Fig. 8B), within which differences between
presynaptic calcium transients cannot be measured with confidence.
Similar correlations measured from five additional preparations are
shown as different symbols. When the signal-to-noise ratio of the
fluorescence transients was favorable, it was possible to identify
measurable
Ca50 when
IPSC50 was as low as 0.2 ms (Fig.
8B,
). The resolution of
Ca50
depends on the level of background noise and the rate of rise of the
calcium transients. This analysis sets the quantitative limits of our experimental resolution. Specifically, when
IPSC50 is larger than 0.5 ms (above dotted
line), one can consistently identify a difference in the half-maximal
points of calcium transients. When the signal-to-noise ratio is
favorable, it is possible to detect measurable
Ca50 with confidence when
IPSC50 is as low as 0.2 ms. This quantitative
information is important when one tries to use fluorescence transients
(
Ca50) to determine whether a given
IPSC50 is due to changes in presynaptic
calcium current or other mechanisms downstream to calcium influx.
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Pre- and postsynaptic events measured from the same varicosities
To evaluate possible errors associated with the mismatch between
IPSC measured from a muscle fiber and the presynaptic
Ca2+ transient monitored from a few varicosities,
we measured Ca2+ transients from a cluster of
varicosities that were under a patch pipette. This configuration
ensured a perfect match between the presynaptic
Ca2+ transient and inhibitory postsynaptic
response. Figure 9A
illustrates a patch recording () obtained in the presence of 1 mM
4-AP and 20 mM TEA, in both the patch pipette and the bath solution.
The arrival of the presynaptic action potential is indicated by the fast transient preceding the inhibitory postsynaptic response (Fig.
9A,
). (The inhibitory postsynaptic response has been
inverted to highlight its close correlation with the fluorescence
transient.) The synaptic delay, measured from the point at which the
fast transient crosses baseline (
) to the onset of IPSC (
) was
1.78 ms. The presynaptic Ca2+ transient
(· · ·) measured simultaneously exhibits a delay of 1.04 ms,
measured from the arrival of the action potential (
) to the onset of
the calcium transient (|). Therefore of the 1.78 ms of synaptic
delay, 1.04 ms can be attributed to the delayed activation of
ICa. The relationship between the
Ca2+ transient and the inhibitory response
recorded from the same varicosities is very similar to that obtained
from IPSC recorded with two-electrode voltage clamp. Traces in Fig.
7B show recordings taken from a different preparation where
IPSC (
) was obtained by two-electrode voltage clamp. The rising phase
of the IPSC does not "cross" the rising phase of the
Ca2+ transient (· · ·) as
the inhibitory response recorded with a patch pipette does. Similar
results were obtained from two additional preparations. This small but
consistent quantitative difference could be attributed to 1)
action potential conduction from proximal to distal varicosities
"smearing" the rising phase of the IPSC recorded from the whole
muscle fiber and 2) limited frequency response of the
two-electrode voltage clamp. Nevertheless, the difference is small and
would only be significant when one tries to perform quantitative curve
fitting.
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DISCUSSION |
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In this report, we explore the properties of fluorescence transients generated by calcium-sensitive dyes at the crayfish neuromuscular junction. Analysis of the relationship between the duration of a presynaptic action potential and the time course of the calcium transient allows us to infer the kinetics of calcium channel opening during an action potential. Comparing the inferences derived from fluorescence transients with previously published kinetic properties of ICa in this preparation provides an independent verification of our interpretation of the calcium transients. In addition, we show that the release process of the inhibitor exhibits a calcium cooperativity of 3, which is consistent with measurements obtained using other techniques. Detailed analysis of the calcium transient and IPSC waveforms shows that the presynaptic calcium transient provides information on the time course of presynaptic calcium influx with a high time resolution. Fluorescence transients reported by calcium-sensitive dyes will be an important tool for analyzing the role of presynaptic calcium channels in synaptic plasticity.
Intraterminal calcium concentration
Thus far we have presented our calcium transient in terms of
F/F instead of estimated concentration.
Because the in vivo affinity of Magnesium Green is unknown in crayfish
terminals (Zhao et al. 1996
), estimates of calcium
concentration based on affinity and fluorescence amplitude may be
misleading (Grynkiewicz et al. 1985
). An empiric
estimate of calcium influx using published results obtained by
ratiometric measurements may be more accurate. It has been shown that
the steady-state calcium level is linearly related to the frequency of
action potentials up to 40 Hz at the crayfish excitor and inhibitor
(Tank et al. 1995
). We have further examined the
linearity up to 200 Hz and found that the steady-state fluorescence
change, after a continuous stimulation of 10 s, deviates from
linearity by <5% at 200 Hz (data not shown, also see
METHODS for data obtained from a stimulus lasting 1 s). Because the steady-state level of
[Ca2+]i was ~1.5 µM
during a 40-Hz train (Tank et al. 1995
), action potential firing at 100 Hz should raise
[Ca2+]i to ~3.8 µM.
Our data showed that the amplitude of the calcium transient activated
by a single broad action potential is on average 29% larger than the
steady-state fluorescence level activated by a 100-Hz stimulation. A
broad action potential should therefore increase
[Ca2+]i to ~4.9 µM.
This estimate is based on the assumption that the amplitude of a
fluorescence transient truly reflects the integration of total calcium
influx. One possible factor that may compromise this assumption is that
fast Ca2+ sequestration processes, such as Na/Ca
exchange, may have started during the relatively long rising phase of
the fluorescence transient activated by a broad action potential. As a
result, the amplitude of the fluorescence transient would represent an
underestimate of total calcium influx. The rate of
Ca2+ sequestration can be inferred from the decay
time constant of the fluorescence transient. The fast component of the
fluorescence transient, following a broad action potential, has an
averaged decay time constant of 70 ± 20 ms (n = 14; see also METHODS for decay time constants elicited by
firing action potentials at 100 Hz in control saline). The average
10-90% rise time of fluorescence transients, measured from the same
14 preparations, is 3.8 ± 0.8 ms. Therefore the decay of a
fluorescence transient is not rapid enough to significantly reduce the
peak amplitude of the fluorescence transient activated by a broad
action potential.
The estimate of the Ca2+
concentration increase that accompanies a broad action potential, 4.9 µM, only reflects a crude approximation of the spatiotemporal
dynamics of presynaptic free calcium. For example, the calcium
transient measured from the central region of a bouton of the lizard
neuromuscular junction shows a detectable difference from that measured
from the edge of the same bouton (David et al. 1997).
The calcium transients recorded from the two regions exhibit different
maximal amplitudes that take ~15 ms to dissipate. Because the
dimensions of the crayfish neuromuscular varicosities are similar to
those of the lizard boutons, ~4 µm diam, it is reasonable to assume
that dissipation of the calcium gradient occurs on a similar time scale
in the crayfish varicosities. Our photometric measurements blend
together fluorescence transients originating from different
compartments without distinction. As a result, although the rising
phase of the calcium transient should be mainly dictated by the time
course of calcium influx, diffusion of calcium ions may also contribute
to the shaping of the rising phase. Therefore the time course of the
presynaptic calcium transient reported here cannot be easily
transformed into that of presynaptic calcium influx (Sabatini
and Regehr 1998
; Sinha et al. 1997
). Nevertheless, our results show that it is possible to clearly identify
circumstances where changes in presynaptic calcium current, as
suggested by differences in IPSC time course, are accompanied by
corresponding shifts in the time course of the fluorescence transient
(Fig. 8).
Time course of calcium influx during an action potential
Comparison of the waveforms of the presynaptic action potential
and the calcium transient indicates that the latter reaches its maximum
when a narrow action potential has nearly repolarized to the level of
resting membrane potential. Because the time course of the calcium
transient roughly approximates an integration of presynaptic calcium
influx (Sabatini and Regehr 1998; Sinha et al.
1997
), the maximal level of a calcium transient should signal the end of calcium influx. Therefore calcium influx should end roughly
when an action potential is repolarized to resting level. This
interpretation is consistent with direct measurements of presynaptic
calcium current in the squid giant synapse (Llinás et al.
1982
; see also Augustine 1990
for simulated
calcium influx) and the calyx of Held (Borst and Sakmann
1998
). In both synapses, the peak of
ICa occurs during the falling phase of
an action potential, and the ICa
terminates roughly when the action potential returns to resting level.
A similar conclusion was derived from a calcium imaging study of the
cerebellar parallel synapse at room temperature (Sabatini and
Regehr 1996
). Finally, our observation that the delay in the
calcium transient is ~1 ms after the onset of an action potential is
also in good agreement with the activation kinetics derived from
ICa measured by voltage clamp
(Wright et al. 1996b
). Therefore the calcium transients
described in this report appear to reflect essential kinetic features
of presynaptic calcium current.
The reason why a calcium transient reaches its maximal level, which
signifies the end of calcium influx, before a broad presynaptic action
potential has fully repolarized is not intuitively obvious. Our
observation is, however, consistent with a recent voltage-clamp study
(Park and Dunlap 1998) that showed that, if a 7.5-ms
action potential was used as a commanding waveform,
ICa returned to baseline when the
action potential repolarized to
25 mV. In the same study, it was
shown that ICa terminated at the same
moment that a narrow action potential repolarized to resting level.
This type of behavior can be attributed to the closing rate of calcium
channels. Specifically, the falling phase of a normal action potential
is rapid in comparison to the rate of channel closing. As a result,
ICa is mainly tail current. However,
the slow repolarization of a broad action potential would mean that the
closing process of Ca channels is constantly in equilibrium. Together
with a slight inactivation of Ca channels (Wright et al.
1996a
), ICa would be fairly
small when a broad action potential had repolarized to the threshold
level for Ca channels. Therefore fluorescence transients activated by
broad action potentials also seem to faithfully track presynaptic
calcium influx.
Two more alternative interpretations of this observation should also be considered. First, given the broad peaks of the calcium transients activated by broad action potentials, identification of the maximal point may not be accurate (see Fig. 6, for example). Second, because the action potentials were recorded at the main branch of the inhibitor while fluorescence transients were measured from varicosities, it is possible that the shape of action potentials differs between the two sites. The latter possibility could best be resolved by measuring presynaptic action potential at the varicosities with voltage-sensitive dyes.
Calcium cooperativity at the inhibitor
Our estimate of calcium cooperativity is similar to those obtained
from direct measurement of presynaptic
ICa (Wright et al. 1996a), from a calcium decaging experiment (Lando and
Zucker 1994
) and those correlating fura-2 estimated presynaptic
calcium concentration and asynchronized release (Ravin et al.
1997
). The consistency of our data with other methods of
estimating calcium cooperativity suggests that the peak level of
fluorescence transients accurately reflects total calcium influx.
Presynaptic calcium current measured by voltage clamp in the
crayfish neuromuscular junction shows that
ICa reaches a steady state ~2 ms
after a voltage step reaches +10 mV (Wright et al. 1996b). This rate of ICa
activation suggests that there could be two phases of increase in
calcium influx as action potential duration is increased. Small
increases in action potential duration, from 0.8 ms up to 2 ms, could
increase calcium influx by increasing the number of open channels and
by increasing channel opening duration. As action potential duration
increased further, beyond 2 ms, the number of open channels would
stabilize, and the increase in calcium influx could best be attributed
to an increase in the duration of channel opening, or repetitive
openings. The two distinct phases of increase in calcium influx should
theoretically result in two separate values of cooperativity.
Specifically, small broadening of an action potential would most likely
increase transmitter release by the "nonoverlapping" domain process
where an increase in release is linearly related to an increase in the
number of open channels (Fogelson and Zucker 1985
;
Simon and Llinas 1985
). This situation is similar to the
finding at the squid giant synapse where an increase in transmitter
release due to broadening of an action potential exhibits a calcium
cooperativity of 1 (Augustine 1990
). As the duration of
an action potential increases further, the probability of simultaneous
opening of neighboring calcium channels increases. The spatial
redistribution of calcium influx would effectively increase the calcium
concentration around vesicles not directly coupled to calcium channels,
by overlapping microdomains (Borst and Sakmann 1996
;
Fogelson and Zucker 1985
; Mintz et al. 1995
; Roberts 1994
; Simon and Llinas
1985
). Calcium cooperativity measured under this condition
typically exhibits a high value. For example, a cooperativity of 3 was
reported at the crayfish excitor when calcium influx was increased by
prolonging the duration of voltage-clamp steps (Wright et al.
1996a
). Increasing the duration of action potentials of the
parallel fiber increases calcium influx mainly by lengthening the
duration of calcium channel opening (Sabatini and Regehr
1997
). The cooperativity measured under these conditions is
also high (3). The logic outlined here would predict two measurable
cooperativities for the crayfish neuromuscular junction, and account
for earlier observations that a cooperativity of 1.5 was observed when
calcium influx was increased by 50% (Delaney et al.
1991
). However, our results show only one cooperativity over
the entire range of calcium influx examined here. It should be noted
that the signal-to-noise ratio of our data are sufficient to resolve a
low cooperativity, if it exists, at the low end of the double logarithm
plot. Furthermore, a cooperativity of ~2.7 (n = 3)
was obtained when we further improved the resolution of our
postsynaptic recording by measuring inhibitory postsynaptic potential
(IPSP), rather than IPSC (Fig. 7,
and
; direct recording of
IPSPs, in the range of
100 µV, provides a better signal-to-noise ratio than corresponding IPSCs recorded with 2-electrode voltage clamp). Therefore, despite the suggestion that different modes of
increasing [Ca2+]i could
occur across the range of calcium influx examined here, we observed
only one cooperativity in this preparation. This apparent contradiction, however, is not unique. For example, calcium
cooperativity measured from ICa
activated by different voltage steps yields only one value for
cooperativity in the squid giant synapse (Augustine and Charlton
1986
; Llinás et al. 1981
), the crayfish
neuromuscular junction (Wright et al. 1996a
), and
chromaffin cells (Engisch and Nowycky 1996
).
Manipulation of calcium influx by voltage-clamp steps, or by broadening
of action potentials, changes the spatial profiles of calcium
concentration at release sites by concurrently varying the number of
open channels and single-channel current. As a result, total calcium
influx monitored by voltage clamp, or by imaging technique, may not
linearly reflect calcium concentration near synaptic vesicles
(Bertram et al. 1996
; Klinauf and Neher 1997
; Roberts 1993
; Winslow et al.
1994
; Yamada and Zucker 1992
). Therefore calcium
cooperativity measured here, and previous voltage-clamp studies, may
have different physiological implications from that obtained by
changing extracellular calcium concentration. Cooperativity, nevertheless, remains a useful parameter for defining characteristics of transmitter release under different conditions.
Time resolution of the fluorescence transients
One of the potential applications for the present experimental
approach is in detecting modulation of presynaptic calcium current as a
possible mechanism underlying synaptic plasticity. To achieve this
goal, one must be able to determine the degree of time resolution that
this experimental method can provide. Data provided in this report
allow us to address this question empirically. Our results show that
one can confidently expect to detect a measurable change in presynaptic
calcium transients if the modulation of calcium channels results in a
shift in IPSC half-maximal points of as little as 0.3 ms. With this
degree of time resolution, one could then begin to determine whether
changes in transmitter release kinetics during F2 facilitation
(Vyshedskiy and Lin 1997a) and during modulator mediated
synaptic enhancement (Vyshedskiy et al. 1998
) are due to
an acceleration in calcium influx.
In conclusion, this report analyzes the use of fluorescence transients generated by calcium-sensitive dyes at the crayfish neuromuscular junction. Our results demonstrate the applicability of this technique to the measurement of cooperativity of transmitter release and to the detection of changes in presynaptic calcium influx.
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ACKNOWLEDGMENTS |
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We thank N. Schweitzer for correcting our English.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-31707 to J.-W. Lin.
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FOOTNOTES |
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Address for reprint requests: J.-W. Lin, Dept. of Biology, Boston University, 5 Cummington St., Boston, MA 02215.
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 17 August 1998; accepted in final form 7 June 1999.
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REFERENCES |
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