|
|
|
|
|
|
|
||
Original Article |
Amplification of Exocytosis and Plasticity
b Department of Physiology, School of Medicine, Nagoya University, Nagoya 466-8550, Japan
Department of Physiology, School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.81-52-744-2049
kubak{at}med.nagoya-u.ac.jp
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials And MethodsResultsDiscussionREFERENCES |
|---|
Key Words: Ca2+-induced Ca2+ release • Ca2+-dependent priming • transmitter release • end-plate potential • frog motor nerve terminals
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials And MethodsResultsDiscussionREFERENCES |
|---|
). A large increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) close to the internal orifice of the Ca2+ channel (Ca2+ microdomain) is thought to activate low affinity Ca2+ receptors of exocytotic machinery (Llinás et al. 1992; Heidelberger et al. 1994; Schweitzer et al. 1995). A global rise in [Ca2+]i in the terminal produced by repetitive nerve activity as well as a rise in the Ca2+ microdomain also regulates the efficacy of impulse-induced exocytosis of neurotransmitter, presumably via high affinity Ca2+ receptors (Kamiya and Zucker 1994; Zucker 1996). These Ca2+ actions on transmitter release processes should be enhanced, if intracellular Ca2+ release occurs in response to Ca2+ entry, similar to the cell soma and dendrites of neurons (Kuba 1980; Friel and Tsien 1992; Hua et al. 1993; Llano et al. 1994; Garaschuk et al. 1997; for review, see Kuba 1994). There is little direct evidence, however, for the involvement of Ca2+ entry-induced Ca2+ release in the impulse-evoked exocytosis of transmitter (Onodera 1973; Smith and Cunnane 1996; Mothét et al. 1998).
Previously, we reported a unique Ca2+-induced Ca2+ release (CICR) mechanism via ryanodine receptors at frog motor nerve terminals. Ca2+ entry produced by repetitive nerve activity primes the mechanism of CICR for activation over a few minutes, and then activates CICR, enhancing the asynchronous release of transmitter. This CICR mechanism is inactivated by further Ca2+ entry, and restored by a short pause of Ca2+ entry, but falls in a deprimed state after a long absence of Ca2+ entry (Narita et al. 1998). We now report here that Ca2+ release via this mechanism is closely coupled with Ca2+ entry through voltage-dependent Ca2+ channels and amplifies the impulse-induced rise in [Ca2+]i, enhancing neurotransmitter exocytosis and the longer modes of its short term plasticity, "augmentation" and "potentiation." This novel role of CICR in the amplification of exocytosis would provide an important basis in general for the mechanism of synaptic plasticity.
|
Materials And Methods |
|---|
|
TopABSTRACTIntroductionMaterials And MethodsResultsDiscussionREFERENCES |
|---|
). Cutaneus pectoris or sartorius muscles of frogs (Rana nigromaculata) were isolated with the innervating nerve. There was no difference in the results obtained from these two types of muscle. The composition of normal Ringer's solution (mM) was: 100 NaCl, 2 KCl, 2.5 CaCl2, 3.0 MgCl2, 8.0 Tris, pH 7.4, or 111 NaCl, 2 KCl, 1.8 CaCl2, 5.0 HEPES-Na, pH 7.4, 5.0 glucose. There was no significant difference in the data obtained from the preparation isolated in different types of Ringer's solution. A conventional intracellular recording technique was applied to record end-plate potentials (EPPs) using electrodes filled with 3 M KCl at 20–24°C. Low Ca2+, high Mg2+ solutions were made by lowering CaCl2 (0.02–0.5 mM) and adding MgCl2 (10 mM) with the adjustment of osmolarity by changing NaCl concentration. The quantal content (QC) of EPP was calculated by dividing the mean amplitude of EPP by the mean amplitude of miniature EPPs (MEPPs) recorded under the same condition. The time course of end-plate current (EPC) was calculated by the deconvolution of that of EPP (digitized at 50 kHz). The Fourier transform of EPP was taken and divided by the Fourier transform of the cable equation (the membrane time constant, 20 ms) for the impulse response (Fatt and Katz 1951; Gage and McBurney 1973). The inverse Fourier transform of the result of division yielded the time course of EPC. The rising phase of the calculated EPC would reliably reflect that of the EPC recorded by voltage clamping the end-plate membrane for two reasons. First, the applicability of the cable equation to the muscle end-plate membrane has been well established (Fatt and Katz 1951; see review by Gage 1976). Second, the time course of the EPC calculated from an EPP was nicely fit to that of the EPC recorded under the voltage-clamp condition (Takeuchi and Takeuchi 1959; see Gage 1976).
Changes in [Ca2+]i in motor nerve terminals were measured from those of frog (Rana nigromaculata) cutaneus pectoris muscles separately from the experiments recording EPPs. The composition of normal Ringer's solution (mM) was: 112 NaCl, 2 KCl, 1.8 CaCl2, 2.4 NaHCO3, pH 7.4, when equilibrated with air, with or without glucose 5.0, or the Ringer's solution buffered with HEPES used for recording EPPs (see above). There was no significant difference between the characteristics of the impulse-induced Ca2+ dynamics and CICR recorded in different types of Ringer's solution. Low Ca2+, high Mg2+ solutions were similar to those for recording EPPs. K-salt of dextran-conjugated Oregon green BAPTA-1 (d-OGB-1: mol wt 10,000) was loaded into the terminals as described previously (Narita et al. 1998). This high-affinity Ca2+ indicator yielded the excellent signal to noise ratio (S/N ratio) of an impulse-induced rise in [Ca2+]i in frog motor nerve terminals. Although we tried to use a low-affinity Ca2+ indicator, OGB-5N, which would be apparently suited for recording high [Ca2+]i in the microdomain for exocytosis at a faster rate, we were unable to record reliably impulse-induced rises in [Ca2+]i at a high S/N ratio with this indicator because of its weak fluorescence before stimulation. In most experiments, a confocal laser scanning unit (MRC-600; Nippon Biorad) with a Kripton-Argon laser (488 nm) in combination with an inverted microscope (TMD-300; objective, Nikon 40x water/N.A. 1.15; Nikon, Inc.) was used to measure fluorescence changes of d-OGB-1. Fluorescence images were taken by a line-scanning mode at 500 Hz. In some experiments, fluorescence was measured with a cooled CCD-camera (Argus/HiSca, C6790-81; Hamamatsu Phototonics) through an image intensifier attached to an upright microscope (Zeiss Axioscope, objective 60x water/N.A. 0.95; Karl Zeiss Japan) and analyzed by image analysis software (Argus; Hamamatsu Phototonics). In the analyses of fluorescence images of d-OGB-1, the ratios of the images during and after nerve stimulation to that before stimulation were taken and analyzed as in previous experiments (Narita et al. 1998). For conversion of fluorescence intensity ratio to [Ca2+]i, the Kd of d-OGB-1 were assumed to be 500 nM and the ratio of the maximum to the minimum fluorescence was 15.0. The basal [Ca2+]i was assumed to be 10–70 nM for the following reasons. When we used the previous value of 72 nM in conversion of some data, the [Ca2+]i value calculated from an increase in fluorescence ratio induced by a single stimulus progressively elevated during a short train under the control condition. We thought that this progressive increase during such a short train was unrealistic, since constant increases in single impulse-induced rise in [Ca2+]i were seen in other experiments such as those at the beginning of a tetanus shown in Fig. 7 C (in which the basal [Ca2+]i was assumed to be 70 nM) and also in other motor nerve terminals (DiGregorio and Vergara 1997; David et al. 1997). Accordingly, we presumed that the apparent, progressive increase in impulse-induced Ca2+ responses seen in some experiments resulted from the incorrect assumption of the basal [Ca2+]i value, which would have been higher than the real one in those terminals.
|
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials And MethodsResultsDiscussionREFERENCES |
|---|
).
|
|
). EPPs slowly and then progressively increased in amplitude during the tetanus, while its failure and variability decreased (Fig. 1C and Fig. D). [When the tetanus was further continued for more than 10 min, the EPP amplitude reached the maximum, and then declined toward the level before the tetanus (not shown) due to the inactivation of CICR, as seen in a MEPP-hump (Narita et al. 1998).]
After this conditioning tetanus, the amplitude and QC of the first EPPs in a short train of 20 stimuli at 50 Hz were markedly enhanced 15.6x (±2.7, SEM, n = 16 for both) those before the conditioning tetanus (0.11 ± 0.02 mV, 0.24 ± 0.05, respectively; Fig. 1 B and 3 A, 
). This enhancement of the first EPP in a short train must be caused by potentiation of transmitter release and the activation of CICR (see below), but not other forms of short-term plasticity, facilitation, and augmentation, because this relatively short-lasting plasticity should have disappeared within a 10-s interval. The decay time course of the enhancement of transmitter release produced by a conditioning tetanus was examined by applying a train of 20 stimuli (50 Hz) every 10 s after the end of the tetanus. The enhancement of the amplitude of the last EPP in a train decayed double exponentially with time constants of 1.85 (±0.15, n = 5: the fraction of amplitude, 48.9 ± 9.2%) and 10.4 (±1.0) min (see Fig. 4 A). The initial component of the enhancement can be explained by potentiation, the longest form of short-term plasticity, since its time constant falls within that of potentiation (Magleby and Zengel 1975). (It may be noted that the enhancement of the last EPP in a short train was multiplied by facilitation, but not by augmentation for the trains given every 10 s.) The time constant of the later component is similar to that of the depriming process of CICR (Narita et al. 1998).
|
and •) and TMB-8 (8 µM: Chiou and Malagodi 1975; not shown). The amplitude and QC of the first EPP in a short train after a conditioning tetanus (10 Hz, 10 min) was 3.2x (±1.7, n = 9 for both) those before the tetanus (0.10 ± 0.04 mV, 0.22 ± 0.08, respectively) in the presence of ryanodine. The amplitude of the last EPP in a short train after the conditioning tetanus (1.02 ± 0.32 mV, n = 6) decayed single exponentially in three cells with time constants (12.8, 15.8, and 3.8 min) or remained unchanged in other cells (Fig. 4 B). This indicates that potentiation as well as the activation of CICR disappeared or decreased markedly in the presence of ryanodine. The slow growth of EPP amplitude during the conditioning tetanus did not continue and changed to a decline before the end of tetanus in the presence of ryanodine (20 µM), indicating that the blockade of ryanodine receptor depends on its activation (Fig. 2C and Fig. D).
|
). The rate of growth of QC of EPPs by a short train was also unchanged by the conditioning tetanus in the presence of ryanodine (Fig. 3 B; x and *). These results indicate that facilitation, presumably the fast component (Zengel and Magleby 1982; Zucker 1996; the fast component comprises 
80%, Tanabe and Kijima 1992), was not affected by the activation of CICR, although it markedly enhanced transmitter release.
Enhancement of Repetitive Impulse-induced Rises in [Ca2+]i by CICR
We examined how the rise in [Ca2+]i in the motor nerve terminal induced by a short train changes after a conditioning tetanus that primes the mechanism of CICR. Rises in [Ca2+]i induced by a short train of stimuli (20–30 stimuli, 50 Hz) were recorded by measuring fluorescence changes of OGB-1 loaded in the motor nerve terminals in a low Ca2+ and high Mg2+ (Ca2+/Mg2+ > 0.2 mM/10 mM) solution with a cooled CCD camera. Changes in the ratio of the fluorescence intensity during and after a short train of stimuli to that before the train were converted to those in [Ca2+]i (Fig. 5 A). A conditioning tetanus (10 Hz, 6 min) caused the marked enhancement of short train-induced rises in [Ca2+]i (to 180 ± 17% at 15–50 s after the tetanus; the control amplitude, 44.9 ± 5.6 nM, n = 7), which lasted for more than 16 min (Fig. 5 A, a). This enhancement was completely blocked by ryanodine (10 µM) applied for 30 min (n = 5; Fig. 5 A, b). Similar enhancements were also seen in normal Ringer's solution (Fig. 5 B; to 166 ± 25% at 10–45 s; the control amplitude, 93.0 ± 11.2 nM, n = 4).
|
|
The long conditioning tetanus thus primes and activates the mechanism of CICR in frog motor nerve terminals (Narita et al. 1998), causing the slow growth of EPP. Once the mechanism of CICR is primed, Ca2+ entry induced by a single nerve impulse markedly enhances neurotransmitter release. Two possible mechanisms may explain the enhancement. First, CICR may amplify the rise in [Ca2+]i in Ca2+ microdomains close to exocytotic machinery so that CICR is directly involved in the impulse-evoked exocytosis. This mechanism may be reflected in the late slow decay phase of the enhanced EPP after the conditioning tetanus (Fig. 4 A). Second, CICR may enhance Ca2+-dependent short-term plasticity of transmitter release, which consists of fast and slow types of facilitation (time constants; 30–60 and 400 ms, respectively), augmentation (7 s) and potentiation (several minutes; Katz and Miledi 1968; Magleby and Zengel 1975; Zengel and Magleby 1982; Delaney and Tank 1994; Regehr et al. 1994; Zucker 1996). Among them, facilitation (presumably fast type) was already shown to be unaffected by the activation of CICR.
Effects of CICR on the Time Courses of Single Impulse-induced Rises in [Ca2+]i, EPP, and End-Plate Current
If the site of CICR is remote from that of Ca2+ entry, CICR should occur with a time delay after Ca2+ entry, and be reflected in the time course of an impulse-induced rise in [Ca2+]i (see DISCUSSION). For instance, if the time delay for Ca2+ release after Ca2+ entry is long enough, the diphasic time course of an impulse-induced rise in [Ca2+]i may be recorded as seen in synaptically evoked Ca2+ transients in the cerebellar Purkinje neurons, in which not only Ca2+ entry, but also inositoltrisphophate-induced Ca2+ release are involved (Finch and Augustine 1998; Takechi et al. 1998). On the other hand, if the time difference between the onsets of Ca2+ entry and Ca2+ release was short enough, a single phasic rise in [Ca2+]i would be seen (see DISCUSSION).
We recorded the whole time course of single impulse-induced rise in [Ca2+]i before and after a conditioning tetanus (10 Hz, 5 min) in normal Ringer. An increase in [Ca2+]i produced by a single stimulus rose in 5 ms (5.7 ± 0.9 ms, n = 6, 10–90% rise time) to the peak (44.7 ± 2.9 nM, n = 6) and decayed double exponentially (
1, 46 ± 6 ms, 79.6 ± 6.4%; 2, 282 ± 70 ms, n = 6; see Fig. 7A and Fig. B). When a conditioning tetanus of 10 Hz for 3–10 min was given to the nerve, a rise in [Ca2+]i induced by individual stimuli slowly increased throughout the course of the tetanus (Fig. 7 C) with the elevation of the basal level by 82 ± 18 nM (n = 5), which decayed with the time constant of 1.65 ± 0.4 min (n = 6: Fig. 7 D). After the conditioning tetanus, impulse-induced rises in [Ca2+]i were markedly enhanced in amplitude (to 3.1 ± 0.7 x the control, n = 7; averaged over those for a period of 5–35 s) and rate of rise (to 3.1 ± 0.8x, n = 6; Fig. 7A and Fig. B). The enhancement decayed over a few minutes to 10 min after the conditioning tetanus (Fig. 7 A). During the enhancement, the peak time was not changed (Fig. 7 B: 105 ± 17% of that before the tetanus, n = 6) and the time constants of the double exponential decay were slightly decreased (1, 88 ± 16%; 2, 79 ± 18%, n = 6) with no change in their fraction (fast component; 70.8 ± 3.9%, n = 6). The enhancement of single impulse-induced rise in [Ca2+]i after the conditioning tetanus was not seen in the presence of thapsigargin applied for 30–90 min (92 ± 11%, n = 4; the magnitude of rise before the conditioning tetanus was 67.8 ± 17.2 nM). Pharmacological priming of the CICR mechanism by caffeine (2 mM) also enhanced single impulse-induced increases in [Ca2+]i (to 160 ± 20%, n = 5; control, 40.3 ± 0.3 nM) with no change in peak time (94 ± 3%; control, 4.1 ± 0.6 ms; Fig. 8A and Fig. B). Thus, the activation of CICR does not apparently prolong the time course of a single impulse-induced rise in [Ca2+]i, but markedly enhances its amplitude.
|
|
|
Changes in [Ca2+]i in the nerve terminal during each repetitive tetani were then observed in experiments separately from those for EPPs. The results were in general similar to those of EPP except for two respects (see below; Fig. 11, A–C). The diphasic rise in [Ca2+]i was seen in all Ca2+ responses. (Its absence in trains of EPPs in the first and second tetanus may be explained by the low probability of transmitter release under this condition.) The declining rate of the second phase of an increased [Ca2+]i during each tetani in the waning phase was faster than that of QC in the corresponding phase (Fig. 11 C). [Augmentation and potentiation of transmitter release developed during tetanus (see below) could explain the slower decay of QC.] Aside from these differences, similar changes in EPP and [Ca2+]i in the nerve terminal during repetition of tetani can be accounted for by the priming and inactivation of CICR (Narita et al. 1998). The waxing and waning of the initial phase of increases in EPP and [Ca2+]i during each tetani reflect the extent of priming and inactivation of CICR, respectively, which modified or produced augmentation and potentiation, while those of the second phase represent the rates of priming and inactivation, respectively, during each tetani.
|
; Zengel and Magleby 1982) and the depriming of CICR, as shown in Fig. 4 A. The magnitudes of these components of QC were increased in the waxing phase of responses during repetition of tetani (Fig. 10 B), and then decreased in the waning phase (Fig. 10 C). The increases of these components of QC in the waxing phase indicate the enhancement of augmentation and potentiation in parallel with the growth of CICR activation, while the decreases in the waning phase may reflect the reduction of augmentation, potentiation, and CICR. The decrease in the second slow component of the increased QC with repetition of tetani in the waning phase (Fig. 10 C), however, was slightly faster than the late decay component of the enhanced EPP after a long conditioning tetanus (Fig. 4 A). Such a faster decrease may also involve other factors, such as depletion of transmitter pool, although it is not large and is equivalent to that caused by stimulation at 3.3 Hz in normal Ringer (33.3 Hz x 20 QC = 3.3 Hz x 200 QC). |
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials And MethodsResultsDiscussionREFERENCES |
|---|
Impulse-induced Ca2+ Dynamics in the Active Zone and Global Cytoplasm
It may be necessary to review briefly the current understanding of impulse-induced Ca2+ dynamics in presynaptic nerve terminals as the basis of interpreting the present findings in terms of the roles of CICR in the exocytosis of transmitter and its short-term plasticity. Ca2+ entry through voltage-dependent Ca2+ channels activated by a nerve impulse increases [Ca2+]i to >100 µM at the active zone and activates exocytosis of transmitter (Katz 1969; Llinás et al. 1992; Heidelberger et al. 1994; Schweitzer et al. 1995). Ca2+ ions in the high [Ca2+]i domain spread throughout the cytoplasm by diffusion, while they are being bound to Ca2+-binding proteins or taken up into organelles (Sala and Hernández-Cruz 1990; Sinha et al. 1997). Thus, most of the increased [Ca2+]i decays within a few milliseconds and the residual component disappears with a time constant of tens of milliseconds (Simon and Llinás 1985; Digregorio et al. 1999; Suzuki et al. 2000). Because of the extremely short lifetime of the high [Ca2+]i and its fast binding and unbinding to the Ca2+-binding proteins of low affinity for the exocytotic machinery, the process of impulse-induced exocytosis is most resistant to the action of fast chelator, BAPTA, among Ca2+-dependent presynaptic processes (Tanabe and Kijima 1992) and unaffected by a slow chelator, EGTA. On the other hand, the [Ca2+]i in the global cytoplasm rises to a moderate level with a slower rate and cleared by several independent mechanisms with time constants of tens of milliseconds, hundreds of milliseconds, and several seconds (Suzuki et al. 2000). The decay time courses of these residual [Ca2+]i in the active zone and global cytoplasm cause various forms of short-term plasticity, fast (time constant, 30–60 ms) and slow (200–400 ms) facilitation, augmentation (several seconds), and potentiation (several tens of seconds).
No Apparent Changes in Facilitation by the Activation of CICR
The high level of residual Ca2+ in the microdomain would produce the fast component of facilitation via the Ca2+-sensing protein for the exocytotic machinery (Katz and Miledi 1968) or a similar one (Kamiya and Zucker 1994; Delaney and Tank 1994), as supported by the following evidence. Fast facilitation is similar in decay time constant to the decay time course of the [Ca2+]i in the microdomain estimated in simulation (Suzuki et al. 2000). Fast facilitation is reported to be most effectively blocked by loading BAPTA (Tanabe and Kijima 1989), but less by EGTA (Suzuki et al. 2000) or uncaging chelators in presynaptic terminals (Kamiya and Zucker 1994), as expected from the involvement of a low-affinity Ca2+ binding protein and the short life time of the high residual [Ca2+]i. Furthermore, the residual [Ca2+]i less than micromolar in the global cytoplasm cannot explain fast facilitation produced via the low affinity Ca2+ receptor (100 µM; see Suzuki et al. 2000).
In the present experiments, fast facilitation of transmitter release produced by a short train of stimuli was not changed after a conditioning tetanus that primes the mechanism of CICR. Since fast facilitation is highly likely caused by the residual [Ca2+]i in the microdomain after a nerve impulse (see above), this absence of the effect of CICR activation on facilitation could be accounted for by two possibilities. First, CICR would occur in a region remote from the active zones so that it does not affect facilitation. In this case, CICR does not directly enhance the impulse-induced exocytosis of transmitter. Second, CICR may amplify the residual [Ca2+]i in the Ca2+ microdomain for exocytosis in proportion to the enhancement of the impulse-induced increase in [Ca2+]i there. (This mode of increases in the residual [Ca2+]i in the microdomain may not be held, however, in normal Ringer solution for the nonlinear binding of Ca2+ to buffers in the terminal.) This implies that CICR takes place in the Ca2+ microdomain for exocytosis so that CICR would directly activate the Ca2+-sensing protein for the exocytotic machinery (see below).
Since the slow component of facilitation was not separated from changes in the amplitude of EPPs induced by a short train, it was not studied how CICR affects this component. It may be possible that slow facilitation could be affected by CICR, similar to augmentation and potentiation for its similar Ca2+ dependence (see below).
Enhancement of Augmentation and Potentiation by the Activation of CICR
The moderate level of residual Ca2+ in the global cytoplasm (and also the microdomain) would activate (or enhance) the mechanisms of slow facilitation (Suzuki et al. 2000; see also Tanabe and Kijima 1992), augmentation, and potentiation (Kamiya and Zucker 1994; Delaney and Tank 1994; Regehr et al. 1994) via a high-affinity Ca2+ receptor. The time courses of augmentation and potentiation (and presumably also slow facilitation) therefore follow the time course of the global rise in [Ca2+]i in the terminal, although augmentation and potentiation would remain longer than the increased [Ca2+]i for their longer lifetime of activation (Regehr et al. 1994). The involvement of high-affinity Ca2+ binding proteins of rather slow binding and unbinding rates in augmentation and potentiation can be suggested by the following observations. Tanabe and Kijima 1989, Tanabe and Kijima 1992 showed little effect of BAPTA on slow facilitation, augmentation, and potentiation at frog motor nerve terminals, while EGTA weakly affected slow facilitation (Suzuki et al. 2000). The rather slow rate (relative to that on fast facilitation) of the appearance of the blocking action of uncaged chelators on augmentation and potentiation was seen at crayfish motor nerve terminals (Kamiya and Zucker 1994).
In the present study, repetition of tetani demonstrated that augmentation and potentiation were enhanced in parallel with the increase in the magnitude of CICR by the priming of CICR. In addition, the initial decay time constant (1.85 min; Fig. 4 A) of enhancement of EPP after a long conditioning tetanus that primed CICR was similar to the decay time constant of a rise in [Ca2+]i induced by a similar conditioning tetanus (1.65 min; Fig. 7 D), and also similar to the time constant calculated from the experimentally derived equation for the decay of potentiation (Magleby and Zengel 1975). This conforms to the role of the residual [Ca2+]i in the global cytoplasm in potentiation (Delaney and Tank 1994; Kamiya and Zucker 1994; Regehr et al. 1994; see above). Furthermore, potentiation of transmitter release by a long conditioning tetanus was completely blocked by ryanodine (Fig. 4). Although it remains to be studied whether the activation of CICR is fully essential for the generation of potentiation, it is quite clear that the largest fraction of potentiation depends on the rise in [Ca2+]i caused by the activation of CICR. This is consistent with the inhibition of potentiation by caffeine (Onodera 1973) and more or less similar to the role of Ca2+ release in potentiation at other terminals, albeit the origins and/or pathways for Ca2+ release are different (Hashimoto et al. 1996; Tang and Zucker 1997).
Involvement of CICR in the Impulse-induced Exocytosis of Transmitter
The question as to whether or not CICR occurs in the Ca2+ microdomains for exocytosis can be examined by analyzing the temporal characteristics of single impulse-induced rise in [Ca2+]i. The impulse-induced rise in [Ca2+]i involving CICR should be the algebraic sum of the time course of the rises in [Ca2+]i produced by Ca2+ entry and CICR. These processes could be obviously hampered to some extent by the rate of Ca2+ binding to Ca2+-binding proteins and Ca2+ probes (Sala and Hernández-Cruz 1990; Sinha et al. 1997), the saturation of Ca2+ probes and the nonlinear dependence of the global [Ca2+]i on the domain [Ca2+]i. Nevertheless, the activation of CICR should be delayed to a Ca2+ entry-induced rise in [Ca2+]i for the time of Ca2+ diffusion from the site of Ca2+ entry to the site of CICR and the rate of the resultant Ca2+ release. This time delay would thus be reflected in the time course of the impulse-induced rise in [Ca2+]i, depending on Ca2+ diffusion path. If CICR occurs at a site close to the site of Ca2+ entry in the active zone, a single impulse-induced increase in [Ca2+]i would rise monophasically with or without slight prolongation of the peak time. If CICR occurs at a site remote enough from that of Ca2+ entry, the time delay for the activation of CICR should be considerably greater and result in diphasic or show double peaks of single impulse-induced rise in [Ca2+]i or at least its prolongation.
There was no change in the peak time of the single impulse-induced rise in [Ca2+]i that was enhanced by a conditioning tetanus (Fig. 7 B). Thus, CICR should occur within the time resolution of 2 ms for [Ca2+]i measurement. Furthermore, if the peak time of the rate of rises in impulse-induced fluorescence is measured with a low affinity Ca2+ indicator (Sabatini and Regehr 1998), this upper bound of 2 ms for the latency of CICR could be much smaller.
The time difference between Ca2+ entry and the resultant Ca2+ release was more relevantly measured by the analysis of the rising phase of EPC, which would reflect the rate of rise of transmitter release and, therefore, that of transmitter exocytosis. The peak time of EPC was increased by 200 µs (Fig. 9 B) after a conditioning tetanus. This prolongation of the time course of impulse-induced exocytosis could reflect the prolongation of the life time of the high [Ca2+]i in the active zone involved in exocytosis. [It is unlikely that the prolongation results from the increased life time of transmitter in the synaptic cleft by the conditioning tetanus (e.g., by inhibiting cholinesterase), since it is unrealistic to assume that presynaptic nerve activity inhibits the enzyme.] The prolongation of the rising phase of EPC together with the enhancements of impulse-induced rise in [Ca2+]i and exocytosis thus suggest the recruitment of additional trigger Ca2+ for exocytosis, which is CICR. In other words, the results strongly indicate the activation of CICR in, or in a region close to, the active zone. The upper bound for the distance (r) between a Ca2+ channel and a ryanodine receptor for Ca2+ diffusion may be estimated from the increase in the peak time (200 µs) of EPC and found to be <109 nm [r = (6 tD)1/2 = 109 nm: D, diffusion coefficient, 10–7 cm2/s; Regehr and Atluri 1995].
There are other lines of evidence for the occurrence of CICR in the active zone of the motor nerve terminals. First, the late decay component of the enhancement of EPPs after a conditioning tetanus slower than that of potentiation must be explained by a new mechanism. The time constant (10.4 min) of this slower decay component of EPP enhancement (Fig. 4 A) is similar to the decay time course of the increase in the single or repetitive impulse(s)-induced rise in [Ca2+]i after a conditioning tetanus (Fig. 5 and Fig. 7) and also to that of the depriming process of CICR (Narita et al. 1998). Second, the mechanism of CICR remained to be inactivated by repetitive Ca2+ entries with a negligibly small elevation of the basal [Ca2+]i in a low Ca2+, high Mg2+ solution, but was quickly restored from inactivation by stopping Ca2+ entry for a few seconds (Narita et al. 1998). This finding indicates the inactivation of ryanodine receptor (Sutko and Airey 1996) by a rise in [Ca2+]i close to the site of Ca2+ entry, and also the activation of CICR in the active zone.
Consequently, it is highly likely that CICR occurs at a site close to the high [Ca2+]i microdomain produced by a nerve impulse and is directly involved in the impulse-induced exocytosis of transmitter. The absence of changes in facilitation of transmitter release during the enhancement of impulse-induced exocytosis then suggests that the impulse-induced rise in [Ca2+]i at the active zone and the residual [Ca2+]i there are proportionally amplified by the activation of CICR (see above). Furthermore, the augmentation and potentiation of EPP induced by a relatively long tetanus in frog motor nerve terminals was little affected by BAPTA (Tanabe and Kijima 1989). This observation could be explained by the occurrence of CICR in the active zone and its enhancement (or activation) of augmentation and potentiation.
The magnitude of the enhancement of exocytosis that attributed to an increase in [Ca2+]i by CICR at the exocytotic sites may be estimated from the fraction of the later decay phase of the enhancement of EPP by a conditioning tetanus. It was 51% of the total enhancement at the end of the tetanus (Fig. 4). The amplification (or generation) of augmentation and potentiation by CICR can explain other components of the enhancement.
Physiological Significance of the Unique CICR Mechanism at Presynaptic Terminals
The physiological significance of the marked enhancement of evoked exocytosis by the unique CICR mechanism at the frog motor nerve terminals lies in its novel mechanisms of priming and activation. The mechanism of priming of CICR is now being studied (Hachisuka et al. 1999). It is at least known that the priming process depends on the rate of nerve stimulation and external Ca2+, indicating the amount of Ca2+ entry (Narita et al. 1998). The loading of Ca2+ into Ca2+ stores in the terminals does not appear to be the mechanism for the priming process (particularly in a low Ca2+, high Mg2+ solution), as already discussed in the previous study (Narita et al. 1998). In brief, the slow rising phase of the conditioning tetanus-induced increase in [Ca2+]i must be caused by Ca2+ entry at the cell membrane and CICR through slowly primed ryanodine receptors. The fraction of the former was found to be much smaller than that of the latter, as evidenced by a small tetanus-induced rise in [Ca2+]i in the presence of a blocker of ryanodine receptors in a low Ca2+, high Mg2+ solution (see Figure 3 of Narita et al. 1998). Accordingly, CICR would play a major role in the conditioning tetanus-induced increase in [Ca2+]i, indicating Ca2+ efflux across the Ca2+ store membranes under this condition (Narita et al. 1998). The priming effect of caffeine on CICR without a conditioning stimulation in both normal (Fig. 8) and low Ca2+, high Mg2+ solutions (our unpublished observations) also rules out the role of Ca2+ loading into Ca2+ stores in the priming process during a conditioning tetanus.
This new, use-dependent mode of modification of transmitter exocytosis is now added to, and/or provides in part a mechanism for some of the well-known short-term plasticity. For a short train of presynaptic nerve activity (e.g., less than tens of impulses) facilitation would play a major role in use-dependent plasticity. Longer lasting activity for more than several seconds significantly produces a global rise in [Ca2+]i in the presynaptic terminals and activates augmentation and potentiation, some, if not all, of which could be boosted by the partial activation of CICR. Much longer lasting activity fully primes the mechanism of CICR and produces the marked enhancement of transmitter release by several tens of times, which lasts for >10 min, until the mechanism of CICR is deprimed. The use-dependent efficacy of impulse-induced exocytosis may thus be expressed by the modification of the multiplicative equation comprising each component of the known short-term plasticity (Magleby and Zengel 1982; Tanabe and Kijima 1992) with the addition of the new component involving CICR.
 |
This unique priming-dependent activation of CICR and its strong facilitatory effects on transmitter release provides in general the important mechanism for the plasticity of synaptic gain. In fact, a similar enhancement of transmitter exocytosis was seen in nicotinic synapses of bullfrog sympathetic ganglia (Takeuchi et al. 1999). If this mechanism exists in central synapses, it could be the basis for the mechanisms of short-term memory and learning. Furthermore, it may settle the controversy over the extent of involvement of CICR in synaptically induced rises in [Ca2+]i in the spines and dendrites of central neurons (Svoboda and Mainen 1999), if the CICR there has a priming process.
Abbreviations used in this paper: CICR, Ca2+-induced Ca2+ release; EPC, end-plate current; EPP, end-plate potential; MEPP, miniature EPP; QC, quantal content.
|
ACKNOWLEDGMENTS |
|---|
This study was supported in part by Grants in Aid for Scientific Research (K. Kuba) from the Japanese Ministry of Education, Science and Culture and a Research Project Grant (K. Narita) from Kawasaki Medical School.
Submitted: 13 December 1999
Revised: 28 February 2000
Accepted: 29 February 2000
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials And MethodsResultsDiscussionREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  X. Wang, M. J. Pinter, and M. M. Rich Ca2+ Dependence of the Binomial Parameters p and n at the Mouse Neuromuscular Junction J Neurophysiol, February 1, 2010; 103(2): 659 - 666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. McNally, V. De Crescenzo, K. E. Fogarty, J. V. Walsh, and J. R. Lemos Individual Calcium Syntillas Do Not Trigger Spontaneous Exocytosis from Nerve Terminals of the Neurohypophysis J. Neurosci., November 11, 2009; 29(45): 14120 - 14126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Behringer, L. D. Leite, N. E. Buchholz, M. G. Keeney, W. J. Pearce, C. K. Vanterpool, S. M. Wilson, and J. N. Buchholz Maturation and long-term hypoxia alters Ca2+-induced Ca2+ release in sheep cerebrovascular sympathetic neurons J Appl Physiol, October 1, 2009; 107(4): 1223 - 1234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Behringer, C. K. Vanterpool, W. J. Pearce, S. M. Wilson, and J. N. Buchholz Advancing Age Alters the Contribution of Calcium Release From Smooth Endoplasmic Reticulum Stores in Superior Cervical Ganglion Cells J Gerontol A Biol Sci Med Sci, February 4, 2009; (2009) gln053v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hou, T. Tamura, and Y. Kidokoro Delayed Synaptic Transmission in Drosophila cacophonynull Embryos J Neurophysiol, November 1, 2008; 100(5): 2833 - 2842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shimizu, M. Fukaya, M. Yamasaki, M. Watanabe, T. Manabe, and H. Kamiya Use-dependent amplification of presynaptic Ca2+ signaling by axonal ryanodine receptors at the hippocampal mossy fiber synapse PNAS, August 19, 2008; 105(33): 11998 - 12003. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Nichols, A. F. Dengler, E. M. Nakagawa, M. Bashkin, B. T. Paul, J. Wu, and G. M. Khan A Constitutive, Transient Receptor Potential-like Ca2+ Influx Pathway in Presynaptic Nerve Endings Independent of Voltage-gated Ca2+ Channels and Na+/Ca2+ Exchange J. Biol. Chem., December 7, 2007; 282(49): 36102 - 36111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sakurai, R. J. Calin-Jageman, and P. S. Katz Potentiation Phase of Spike Timing-Dependent Neuromodulation by a Serotonergic Interneuron Involves an Increase in the Fraction of Transmitter Release J Neurophysiol, October 1, 2007; 98(4): 1975 - 1987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kenet, R. C. Froemke, C. E. Schreiner, I. N. Pessah, and M. M. Merzenich Perinatal exposure to a noncoplanar polychlorinated biphenyl alters tonotopy, receptive fields, and plasticity in rat primary auditory cortex PNAS, May 1, 2007; 104(18): 7646 - 7651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Nunez, L. Senovilla, S. Sanz-Blasco, P. Chamero, M. T. Alonso, C. Villalobos, and J. Garcia-Sancho Bioluminescence imaging of mitochondrial Ca2+ dynamics in soma and neurites of individual adult mouse sympathetic neurons J. Physiol., April 15, 2007; 580(2): 385 - 395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Gover, T. H. V. Moreira, J. P. Y. Kao, and D. Weinreich Calcium regulation in individual peripheral sensory nerve terminals of the rat J. Physiol., January 15, 2007; 578(2): 481 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Jackson and S. A. Thayer Mitochondrial Modulation of Ca2+-Induced Ca2+-Release in Rat Sensory Neurons J Neurophysiol, September 1, 2006; 96(3): 1093 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zissimopoulos, D. J. West, A. J. Williams, and F. A. Lai Ryanodine receptor interaction with the SNARE-associated protein snapin J. Cell Sci., June 1, 2006; 119(11): 2386 - 2397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Pagani, R. C. Reisin, and O. D. Uchitel Calcium signaling pathways mediating synaptic potentiation triggered by amyotrophic lateral sclerosis IgG in motor nerve terminals. J. Neurosci., March 8, 2006; 26(10): 2661 - 2672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Ghatpande, K. Sivaraaman, and S. Vijayaraghavan Store Calcium Mediates Cholinergic Effects on mIPSCs in the Rat Main Olfactory Bulb J Neurophysiol, March 1, 2006; 95(3): 1345 - 1355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. R. J. Gordon and J. S. Bains Noradrenaline Triggers Multivesicular Release at Glutamatergic Synapses in the Hypothalamus J. Neurosci., December 7, 2005; 25(49): 11385 - 11395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Liu, B. Chen, M. Yankova, D. K. Morest, E. Maryon, A. R. Hand, M. L. Nonet, and Z.-W. Wang Presynaptic Ryanodine Receptors Are Required for Normal Quantal Size at the Caenorhabditis elegans Neuromuscular Junction J. Neurosci., July 20, 2005; 25(29): 6745 - 6754. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Verkhratsky Physiology and Pathophysiology of the Calcium Store in the Endoplasmic Reticulum of Neurons Physiol Rev, January 1, 2005; 85(1): 201 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. K. Unni, S. S. Zakharenko, L. Zablow, A. J. DeCostanzo, and S. A. Siegelbaum Calcium Release from Presynaptic Ryanodine-Sensitive Stores Is Required for Long-Term Depression at Hippocampal CA3-CA3 Pyramidal Neuron Synapses J. Neurosci., October 27, 2004; 24(43): 9612 - 9622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tully and S. N. Treistman Distinct Intracellular Calcium Profiles Following Influx Through N- Versus L-Type Calcium Channels: Role of Ca2+-Induced Ca2+ Release J Neurophysiol, July 1, 2004; 92(1): 135 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Jin and R. D. Hawkins Presynaptic and Postsynaptic Mechanisms of a Novel Form of Homosynaptic Potentiation at Aplysia Sensory-Motor Neuron Synapses J. Neurosci., August 13, 2003; 23(19): 7288 - 7297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lelli, P. Perin, M. Martini, C. D. Ciubotaru, I. Prigioni, P. Valli, M. L. Rossi, and F. Mammano Presynaptic Calcium Stores Modulate Afferent Release in Vestibular Hair Cells J. Neurosci., July 30, 2003; 23(17): 6894 - 6903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. David and E. F Barrett Mitochondrial Ca2+ uptake prevents desynchronization of quantal release and minimizes depletion during repetitive stimulation of mouse motor nerve terminals J. Physiol., April 15, 2003; 548(2): 425 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Krizaj, F Anthony Lai, and D. R Copenhagen Ryanodine stores and calcium regulation in the inner segments of salamander rods and cones J. Physiol., March 15, 2003; 547(3): 761 - 774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Kang and G. G Holz Amplification of exocytosis by Ca2+-induced Ca2+ release in INS-1 pancreatic {beta} cells J. Physiol., January 1, 2003; 546(1): 175 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R L Simkus and C. Stricker The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex J. Physiol., December 1, 2002; 545(2): 521 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sukhareva, S. V. Smith, D. Maric, and J. L. Barker Functional Properties of Ryanodine Receptors in Hippocampal Neurons Change During Early Differentiation in Culture J Neurophysiol, September 1, 2002; 88(3): 1077 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-F. Lu and R. D. Hawkins Ryanodine Receptors Contribute to cGMP-Induced Late-Phase LTP and CREB Phosphorylation in the Hippocampus J Neurophysiol, September 1, 2002; 88(3): 1270 - 1278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-M. Lu and K. Kuba Synchronous and Asynchronous Exocytosis Induced by Subthreshold High K+ at Cs+-Loaded Terminals of Rat Hippocampal Neurons J Neurophysiol, March 1, 2002; 87(3): 1222 - 1233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J Kennedy and R. W Meech Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+-induced Ca2+ release J. Physiol., February 15, 2002; 539(1): 15 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Carter, K. E. Vogt, K. A. Foster, and W. G. Regehr Assessing the Role of Calcium-Induced Calcium Release in Short-Term Presynaptic Plasticity at Excitatory Central Synapses J. Neurosci., January 1, 2002; 22(1): 21 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Kang, O. G Chepurny, and G. G Holz cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic {beta}-cells J. Physiol., October 15, 2001; 536(2): 375 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Brailoiu, M. D. Miyamoto, and N. J. Dun Nicotinic Acid Adenine Dinucleotide Phosphate Enhances Quantal Neurosecretion at the Frog Neuromuscular Junction: Possible Action on Synaptic Vesicles in the Releasable Pool Mol. Pharmacol., October 1, 2001; 60(4): 718 - 724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Albrecht, S. L. Colegrove, J. Hongpaisan, N. B. Pivovarova, S. B. Andrews, and D. D. Friel Multiple Modes of Calcium-Induced Calcium Release in Sympathetic Neurons I: Attenuation of Endoplasmic Reticulum Ca2+ Accumulation at Low [Ca2+]i during Weak Depolarization J. Gen. Physiol., July 1, 2001; 118(1): 83 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Akita and K. Kuba Functional Triads Consisting of Ryanodine Receptors, Ca2+ Channels, and Ca2+-Activated K+ Channels in Bullfrog Sympathetic Neurons: Plastic Modulation of Action Potential J. Gen. Physiol., November 1, 2000; 116(5): 697 - 720. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
