Correspondence to: K. Kuba, Department of Physiology, School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax:81-52-744-2049 E-mail:kubak{at}med.nagoya-u.ac.jp.
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Abstract |
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Ca2+-induced Ca2+ release (CICR) enhances a variety of cellular Ca2+ signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca2+ entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca2+ entry inactivates the CICR mechanism and the absence of Ca2+ entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (1020 Hz, 210 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca2+ in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca2+ during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca2+ channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca2+-dependent priming.
Key Words: Ca2+-induced Ca2+ release, Ca2+-dependent priming, transmitter release, end-plate potential, frog motor nerve terminals
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INTRODUCTION |
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A nerve impulse triggers exocytosis of neurotransmitter via Ca2+ entry through voltage-dependent Ca2+ channels at the presynaptic terminals (see
Previously, we reported a unique Ca2+-induced Ca2+ release (CICR)1 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 (
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MATERIALS AND METHODS |
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Preparations and experimental procedures are essentially similar to those of the previous study (
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 (
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d-OGB-1 was obtained from Molecular Probes, Inc. TMB-8 [8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride] was from Sigma Chemical Co. or Tokyo Kasei Kogyo Co. Ryanodine and HEPES-Na or -K, were from Sigma Chemical Co.
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RESULTS |
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Enhancement of Impulse-induced Transmitter Release by CICR
EPPs were intracellularly recorded in a low Ca2+, high Mg2+ solution. A short train of 20 stimuli at 50 Hz to the nerve was repeated 1020x every 20 s in a low Ca2+, high Mg2+ solution (Fig 1 A). The averages for the amplitudes of individual EPPs in trains were taken and divided by MEPPs, yielding QC of EPP (see Fig 3 A; and x). The amplitude and QC increased during a short train of stimuli, indicating the facilitation of evoked release of transmitter (
To observe how CICR affects impulse-induced exocytosis, the nerve was stimulated by a relatively long tetanus at a moderate frequency (10 Hz) for 10 min, which is expected to fully prime the mechanism of CICR (
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 (
The enhancement of EPP by a conditioning tetanus was strongly inhibited after treatment with blockers of ryanodine receptors, ryanodine (20 µM: Fig 2 B and 3 B, and ) and TMB-8 (8 µM:
CICR Does Not Affect Facilitation of Transmitter Release
The rate of growth of QC of EPPs induced by a short train of stimuli remained unchanged after a long conditioning tetanus that primed the mechanism of CICR (Fig 3 A; x and ). 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 (
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 (~2030 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 1550 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 1045 s; the control amplitude, 93.0 ± 11.2 nM, n = 4).
To observe further how the increase in [Ca2+]i induced by each impulse in a short train is affected by a conditioning tetanus that primes CICR, line scanning was made to motor nerve terminals loaded with dOGB-1 (Fig 6A and Fig B) in normal Ringer's solution, in which individual rises in [Ca2+]i evoked by each impulse were discerned. Changes in the ratio of the fluorescence intensity during and after a short tetanus to that before the tetanus were converted to those of [Ca2+]i, and then plotted against time (Fig 6 C). Each stimulus during a short train of 20 stimuli (50 Hz) produced rises in [Ca2+]i in the motor nerve terminal, which progressively increased in decay rate and summed up to a plateau within 1015 stimuli for matching of individual decay rates with the stimulation interval (Fig 6 C). The magnitude of the first response in a train was 47.9 ± 4.4 nM (n = 5). The peak of the last response measured from the prestimulation level was 363 ± 42 nM (n = 5). The decay phase of [Ca2+]i after the end of a train followed a double exponential function with time constants, 48 ± 10 ms (59 ± 6% in amplitude) and 410 ± 107 ms (n = 5).
After the application of a conditioning tetanus (20 Hz, 3 min; see changes in [Ca2+]i produced by a similar tetanus in Fig 7 C), a rise in [Ca2+]i induced by each stimulus of a short train (50 Hz, 20 pulses) was amplified (Fig 6B and Fig C; 77 ± 19 nM, n = 5, averaged over the first responses in a short train recorded for 15 s to 3 min). The magnitude of the last responses in a train was 475 ± 68 nM (n = 5), and decayed double exponentially with the time constants (51 ± 8 and 305 ± 43 ms, n = 5; fast component, 52 ± 5%) that were slightly smaller than those before the conditioning tetanus. This amplification in tetanus-induced rises in [Ca2+]i by the conditioning tetanus decayed over a few minutes to >10 min (not shown, but see Fig 5 B for similar experiments).
The long conditioning tetanus thus primes and activates the mechanism of CICR in frog motor nerve terminals (
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 (
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, 1090% 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 310 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 535 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 3090 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.
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The more straightforward way of testing the involvement of CICR in the Ca2+ microdomain for impulse-induced exocytosis would be to examine the time course of transmitter release induced by a nerve impulse. Changes in the time course of transmitter release may be reflected in the rising phase of the EPC underlying an EPP. The peak times of EPP (1.71 ± 0.08 ms, n = 5) and EPC (see MATERIALS AND METHODS for estimation: 596 ± 47 µs) were increased by 227 µs (±69 µs; Fig 9 A) and 200 µs (±65 µs; Fig 9 B), respectively, after the conditioning tetanic stimulation (10 Hz, 10 min) that increased their amplitude markedly. This indicates that the time course of transmitter release is prolonged when CICR is activated in response to Ca2+ entry. This implies that the conditioning tetanus recruited a new impulse-induced source of Ca2+. The extent of the prolongation, however, was not large, suggesting that CICR occurs in a region close to the Ca2+ microdomain produced by Ca2+ entry and contributes directly to impulse-induced exocytosis (see DISCUSSION).
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Enhancement of Transmitter Exocytosis in Parallel with the Extent of the Activation of CICR
How the use-dependent changes of transmitter release are affected by the extent of activation of CICR was examined by observing changes in EPPs and [Ca2+]i in the terminals in response to repetitive tetani. Effects of combinations of a tetanus of 33.3 or 50 Hz and a period of a low rate stimuli (0.5 or 1 Hz), each for 30 s, were first observed on EPPs in a low Ca2+, high Mg2+ solution (Fig 10 A). The amplitude of EPPs induced by each tetani showed complex patterns of changes during the course of tetani. It is, however, clear in overall that the amplitude of EPPs initially increased (waxing phase), reached the maximum, and then declined with the repetition of tetani (waning phase; Fig 10 A). Detailed analyses and consideration of changes in EPPs in response to each tetani revealed changes in the activity-dependent modification of transmitter release during the course of a slow priming, subsequent activation, and inactivation of CICR.
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Fig 10 (B and C) shows changes in QC of EPP during and after each repetitive high frequency tetani. QC was monotonously increased during the first and second tetanus (Fig 10 B). Increases in QC during each subsequent tetani were diphasic; the initial rapid rise and the subsequent slow rise (or decline). The magnitude and rate of the initial growth phase of QC increased with repetition of tetani until the maximum response was reached (in the waxing phase; Fig 10 B). Then, the magnitude of the initial phase decreased with a slight reduction in rate of rise in the later tetani (in the waning phase; Fig 10 C). The rate of rise of the second phase during a tetanus was unchanged in the third to fifth tetani and became plateau in the sixth tetanus (Fig 10 B). The second phase then changed to a declining phase for tetani applied after the maximum response was reached (in the waning phase; Fig 10 C).
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, AC). 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 (
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Enhancement of Augmentation and Potentiation in Parallel with the Extent of CICR Activation
Changes in augmentation and potentiation induced by repetition of tetani are shown by the decay of increased QC of EPPs (induced at 0.5 or 1 Hz) after each tetani. The enhancement of QC declined in two phases with time constants of about a few and several tens of seconds (the precise measurement was not possible for the sparse time resolution; Fig 10B and Fig C, insets). The initial fast phase would represent the decay of augmentation. The second slow phase, which was almost sustained as repetition of tetani proceeded, could be due to the decay of potentiation (
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DISCUSSION |
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The present study demonstrates that the conditioning tetanus to the nerve for priming the mechanism of CICR produced the marked enhancement of the amplitude and QC of end-plate potentials, which was blocked by inhibitors of ryanodine receptors. The enhancement decayed in two phases with the time constants of 1.85 and 10 min, which correspond to those of the use-dependent potentiation of transmitter release and the depriming rate of CICR, respectively, and accompanied by a slight prolongation of the peak times of EPP and EPC. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca2+ in the terminals with little change in time course. Facilitation of EPPs induced by a short train was not changed after the conditioning stimulation, while augmentation and potentiation of EPP induced by a high frequency long tetanus were enhanced by repetition of long tetani that slowly primed CICR. It is discussed below how these results support the hypothesis that ryanodine receptors exist close to voltage-gated Ca2+ channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after the Ca2+-dependent priming.
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 (
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 (
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 (
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 (
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 (
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 (
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;
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 (
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 (
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 (
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 (
where F1, F2, A, P, and R are the fraction of increases produced by fast and slow facilitation, augmentation, potentiation, and CICR, respectively. As already indicated, the increases in [Ca2+]i involved in augmentation and potentiation, especially the latter, are strongly amplified by the activation of CICR at the frog motor nerve terminals.
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 (
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Footnotes |
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Dr. Akita and Mr. Hachisuka contributed equally to this paper.
1 Abbreviations used in this paper: CICR, Ca2+-induced Ca2+ release; EPC, end-plate current; EPP, end-plate potential; MEPP, miniature EPP; QC, quantal content.
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Acknowledgements |
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We thank Prof. H. Kijima for critical comments to the earlier version of the manuscript and Dr. T. Takai for kind computation of EPC.
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
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