Presynaptic Ca2+ Influx at the Inhibitor of the Crayfish Neuromuscular Junction: A Photometric Study at a High Time Resolution

Andrey Vyshedskiy and Jen-Wei Lin

Department of Biology, Boston University, Boston, Massachusetts 02215


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, omega -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega 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 MOmega , 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 MOmega . 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 Delta 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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Fig. 1. Calcium transients recorded from different parts of the inhibitor. The inhibitor was activated by 20 action potentials at 100 Hz, indicated by the horizontal line above the traces. Fluorescence transients, from Magnesium Green, activated by the stimulation were recorded from areas identified in the inset. The calcium transient recorded from varicosities, area 1, exhibits the largest amplitude and fastest decay (numerical labels associated with individual traces correspond to the numbered areas in the inset). The transient recorded from the branch point, area 2, exhibits a much slower decay. The fluorescence change measured from the main axon of the inhibitor, area 3, rises barely above the background noise. Area 4 does not contain any structures from the inhibitor and shows no signal associated with the stimulation. Each trace represents the average of 50 trials. The illustration in the inset is not drawn to scale.

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 (down-triangle) and -2 (triangle ) exhibit a decline in their amplitudes. However, low-affinity dyes, Magnesium Green (open circle ) and Oregon Green 488 BAPTA-5N (), do not show such decline. The saturating behavior of Calcium Green-5N (diamond ), 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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Fig. 2. Saturation of calcium-sensitive dyes. A, left panel: fluorescence transients, from Calcium Green-1, reflecting calcium influx activated by 20, 40, 60, 80, and 100 action potentials delivered at 100 Hz. Horizontal lines at the bottom indicate the duration of stimulation. Right panel: fluorescence transients activated by consecutive bursts of 20 action potentials calculated from traces shown in the left panel. These traces were obtained by subtracting transients activated by n action potentials from those activated by n + 20 action potentials. The progressively decreasing amplitude of the fluorescence transients suggests a saturation of Calcium Green-1. B, left panel: fluorescence transients, reported by Magnesium Green, activated by the same protocol as that used in A. Right panel: fluorescence transients activated by consecutive bursts of 20 action potentials show no sign of saturation. C, left panel: fluorescence transients, reported by Magnesium Green, activated by action potentials at 100 Hz (---), 200 Hz (· · ·) in control saline and by a single and broad action potential (down-arrow ) in 1 mM 4-AP and 20 mM TEA. The amplitude of the latter falls between the maximal fluorescence changes activated by 100 and 200 Hz stimulation. Right panel: fluorescence transients activated by consecutive bursts of 40 action potentials, at 200 Hz, show no sign of saturation. D: summary of dye saturation tested in the crayfish inhibitor. All the tests were performed by the same protocol shown in A-C. Burst number therefore indicates fluorescence transients activated by consecutive bursts of 20 action potentials. Amplitudes of fluorescence transients were normalized to that activated by the 1st burst. , data collected from the 200-Hz protocols discussed in C. Therefore in this case, the burst number refers to the fluorescence transients activated by bursts of 40 action potentials. The sample size for Oregon Green 488 BAPTA-5N is 3, for Magnesium Green at 100 Hz is 5, and at 200 Hz is 6, for Calcium Green-1 is 3, for Calcium Green-2 is 1, for Calcium Green-5N is 2. Error bars represent SD. All traces represent the average of 200 trials.

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 (down-arrow ) 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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Fig. 3. Consistency of calcium transients recorded from different clusters of varicosities. A: calcium transients recorded from areas identified in the inset. Areas 1 and 2 are located on the same muscle fiber but originate from separate secondary branches. The muscle fiber is outlined by dotted lines. Area 3 identifies a large bouton, on a different muscle fiber, with a diameter of 15 µm. Amplitudes of the transients have been normalized to 1. Their original amplitudes were (in %): area 1, 3.09; area 2, 3.24; area 3, 3.39. B: same traces as in A displayed on a fast time scale. The rising phases of the calcium transients are identical, although the decay time constant of trace 3 is significantly slower. Therefore the rising phase of calcium transients recorded from a cluster of varicosities is representative of presynaptic calcium transients. All traces represent the average of 400 trials. The distance between areas 1 and 2 is 250 µm. Inset is not drawn to scale.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 4. Increase in the amplitude of the presynaptic Ca2+ transient as the duration of action potential increases. Top traces: presynaptic transients (A1-A3). Bottom traces: action potentials recorded simultaneously (B1-B3). Trace styles are matched. A1 and B1: recordings obtained in control saline (---), in the presence of 1 mM 4-AP (· · ·), and after the addition of 1 mM TEA to 1 mM 4-AP (- - -). Higher concentrations of TEA were added in the presence of 1 mM 4-AP. A2 and B2: recordings obtained in 2 mM (---) and 4 mM (· · ·) TEA. A3 and B3: recordings obtained in 10 mM (---) and 20 mM (· · ·) TEA. Traces in A1 and B1 are averages of 1,200 trials, traces in A2 and A3 and B2 and B3 are averages of 400 trials. Vertical lines in A1 and A3, and B1 and B3 are used to identify the time at which the presynaptic Ca2+ transients reach their maximal points. All traces share the same time scale.

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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Fig. 5. Summary of the effects of potassium channel blockers on action potential duration (A) and the correlation between action potential duration and presynaptic calcium transient (B). A: action potential duration, measured at -30 mV, increased with progressively advanced block of potassium channels. 4-Aminopyridine (4-AP; 1 mM) was introduced first and remained in the saline through subsequent addition of TEA. Action potential duration measured in control saline is defined as 100%. Each data point is an average obtained from 8 preparations, and error bars represent SD. The data point marked by * was obtained before the effect of 4 mM TEA had reached a steady state. The curve is drawn by hand. B: correlation between the duration of the presynaptic action potential and the amplitude of the calcium transient. Ca2+ transients were measured at their maximal amplitude. All data points were normalized to values measured in control saline. Data collected from each preparation are represented by different symbols. The straight line has a slope of 1.2.

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, open circle ). 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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Fig. 6. Simultaneous recordings of presynaptic action potentials, Ca2+ transients and inhibitory postsynaptic current (IPSC). IPSCs (A1-A3) were recorded with 2-electrode voltage clamp. The duration of the presynaptic action potential (C1-C3) was progressively increased by increasing the level of potassium channel block, which leads to a corresponding increase in calcium transient amplitude (B1-B3) and IPSC (A1-A3). Trace styles are matched. Traces in the left column represent averages of 1,200 trials. Traces in the middle and right columns are averages of 400 trials. All traces share the same time scale. Note that IPSCs in A1 have a different vertical scale.

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, open circle  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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Fig. 7. Calcium cooperativity of the inhibitor. The x-axis represents the maximal amplitude of the calcium transient, normalized to the value measured in control saline. IPSCarea was obtained by integrating IPSC, also normalized to the value measured in control saline. In cases where the action potential fired repetitively, the IPSC associated with the additional spike was replaced by an exponentially decaying time course extrapolated from the initial decay. Results obtained from each preparation are represented by a different symbol.  and , derived from experiments where inhibitory postsynaptic potentials (IPSPs), rather than IPSCs, were used to monitor transmitter release. In these cases, IPSP amplitude, rather than area, was used in the plot. Dotted line has a slope of 2.7 and is drawn to fit the open circles. Solid lines represent cooperativities of 2 and 4, respectively.

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 (Delta IPSC50) is plotted against the corresponding difference for calcium transients (Delta 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 Delta Ca50 when Delta IPSC50 was as low as 0.2 ms (Fig. 8B, triangle ). The resolution of Delta 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 Delta 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 Delta Ca50 with confidence when Delta IPSC50 is as low as 0.2 ms. This quantitative information is important when one tries to use fluorescence transients (Delta Ca50) to determine whether a given Delta IPSC50 is due to changes in presynaptic calcium current or other mechanisms downstream to calcium influx.



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Fig. 8. Resolution of presynaptic calcium transients. A: normalized IPSC, top traces, and presynaptic calcium transients, bottom traces. Differences in the rising phase of adjacent IPSCs can be clearly matched to corresponding differences in the rising phase of calcium transients. The only exception is that the difference between yellow and red calcium transients cannot be separated with confidence, although their corresponding IPSCs exhibit a clearly separated rising phase. Matched traces are represented by the same colors. Changes in the rising phases of IPSC and Ca2+ transients are quantitated by measuring the time difference between their half-maximal points, numbers 1-4. The upswings (up-arrow ) are due to repetitive firing of action potentials. B: correlation between the difference in the half-maximal points between adjacent IPSCs (Delta IPSC50) and that of adjacent Ca2+ transients (Delta Ca50). Squares with enclosed numbers were measured from the traces shown in A, and the numbers match Delta Ca50 and Delta IPSC50 identified in A. Data obtained from 5 additional preparations are also shown in different symbols. Data points within the elongated circle were measured from conditions where Delta Ca50 could not be measured with confidence but where the corresponding difference in IPSC was clear. Data points outside the circle are positively correlated. Data points above the dotted line were obtained from trace pairs that consistently provided clearly measurable Delta Ca50.

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, down-arrow ). (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 (down-arrow ) to the onset of IPSC (up-arrow ) 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 (down-arrow ) 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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Fig. 9. Matched Ca2+ transient and IPSC measured by macropatch recordings. A: presynaptic Ca2+ transient was measured from the same cluster of varicosities as that from which the inhibitory postsynaptic response was measured by a patch pipette. The polarity of the inhibitory response has been inverted. The arrival of the presynaptic action potential is indicated by the biphasic transient (down-arrow ) where the downward portion has been erased. The point where the bipolar transient crosses the baseline is used as the point from which synaptic delay is measured. Upward arrow indicates the point at which the inhibitory postsynaptic response starts. Vertical line identifies the moment when the Ca2+ transient starts. Driving force for the inhibitory response was increased by a hyperpolarizing current pulse injected into the muscle fiber on which these recordings were obtained. The rising phase of IPSC is very fast such that it crosses the Ca2+ transient. Both traces are averages of 400 trials. B: Ca2+ transient and IPSC recorded with 2-electrode voltage clamp from a different preparation. Traces are identical to the dashed traces shown in Fig. 6, A3 and B3. The comparison between A and B shows that IPSC recorded by 2-electrode voltage clamp is slower in that it does not "catch up" with the Ca2+ transient on its rising phase. Both traces in B are averages of 400 trials.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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