Biomedical subjects

T Sakaba

Publications and source records attributed to T Sakaba.

10 recordsLinked to original sources

PubMed2001

Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse.

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high-frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin-dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles.

Action Potentials↗

PubMed2001

Estimating transmitter release rates from postsynaptic current fluctuations.

A method is presented that allows one to estimate transmitter release rates from fluctuations of postsynaptic current records under conditions of stationary or slowly varying release. For experimental applications, we used the calyx of Held, a glutamatergic synapse, in which "residual current," i.e., current attributable to residual glutamate in the synaptic cleft, is present. For a characterization of synaptic transmission, several postsynaptic parameters, such as the mean amplitude of the miniature postsynaptic current and an apparent single channel conductance, have to be known. These were obtained by evaluating variance and two more higher moments of the current fluctuations. In agreement with Fesce et al. (1986), we found both by simulations and by analyzing experimental records that high-pass filtering of postsynaptic currents renders the estimates remarkably tolerant against nonstationarities. We also found that release rates and postsynaptic parameters can be reliably obtained when release rates are low ( approximately 10 events/msec). Furthermore, during a long-lasting stimulus, the transmitter release at the calyx of Held was found to decay to a low, stationary rate of 10 events/msec after depletion of the "releasable pool" of synaptic vesicles. This stationary release rate is compatible with the expected rate of recruitment of new vesicles to the release-ready pool of vesicles. MiniatureEPSC (mEPSC) size is estimated to be similar to the value of spontaneously occurring mEPSC under this condition.

Animals↗

PubMed2001

Combining deconvolution and noise analysis for the estimation of transmitter release rates at the calyx of held.

The deconvolution method has been used in the past to estimate release rates of synaptic vesicles, but it cannot be applied to synapses where nonlinear interactions of quanta occur. We have extended this method to take into account a nonlinear current component resulting from the delayed clearance of glutamate from the synaptic cleft. We applied it to the calyx of Held and verified the important assumption of constant miniature EPSC (mEPSC) size by combining deconvolution with a variant of nonstationary fluctuation analysis. We found that amplitudes of mEPSCs decreased strongly after extended synaptic activity. Cyclothiazide (CTZ), an inhibitor of glutamate receptor desensitization, eliminated this reduction, suggesting that postsynaptic receptor desensitization occurs during strong synaptic activity at the calyx of Held. Constant mEPSC sizes could be obtained in the presence of CTZ and kynurenic acid (Kyn), a low-affinity blocker of AMPA-receptor channels. CTZ and Kyn prevented postsynaptic receptor desensitization and saturation and also minimized voltage-clamp errors. Therefore, we conclude that in the presence of these drugs, release rates at the calyx of Held can be reliably estimated over a wide range of conditions. Moreover, the method presented should provide a convenient way to study the kinetics of transmitter release at other synapses.

Animals↗

PubMed2001

Quantitative relationship between transmitter release and calcium current at the calyx of held synapse.

A newly developed deconvolution method (Neher and Sakaba, 2001) allowed us to resolve the time course of neurotransmitter release at the calyx of Held synapse and to quantify some basic aspects of transmitter release. First, we identified a readily releasable pool (RRP) of synaptic vesicles. We found that the size of the RRP, when tested with trains of strong stimuli, was constant regardless of the exact stimulus patterns, if stimuli were confined to a time interval of approximately 60 msec. For longer-lasting stimulus patterns, recruitment of new vesicles to the RRP made a substantial contribution to the total release. Second, the cooperativity of transmitter release as a function of Ca(2+) current was estimated to be 3-4, which confirmed previous results (Borst and Sakmann, 1999; Wu et al., 1999). Third, an initial small Ca(2+) influx increased the efficiency of Ca(2+) currents in subsequent transmitter release. This type of facilitation was blocked by a high concentration of EGTA (0.5 mm). Fourth, the release rates of synaptic vesicles at this synapse turned out to be heterogeneous: once a highly Ca(2+)-sensitive population of vesicles was consumed, the remaining vesicles released at lower rates. These components of release were more clearly separated in the presence of 0.5 mm EGTA, which prevented the buildup of residual Ca(2+). Conversely, raising the extracellular Ca(2+) concentration facilitated the slower population such that its release characteristics became more similar to those of the faster population under standard conditions. Heterogeneous release probabilities are expected to support the maintenance of synaptic transmission during high-frequency stimulation.

Animals↗

PubMed2001

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held.

We have studied the effects of cAMP on synaptic transmission at the calyx of Held and found that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analog of cAMP) potentiated excitatory postsynaptic currents (EPSCs). Direct sampling of miniature EPSCs (mEPSCs) and nonstationary fluctuation analysis showed that mEPSCs were not modulated by cAMP, suggesting that the locus of modulation is presynaptic. Deconvolution was used to examine effects of cAMP on quantal-release rates. By using this method, it was shown recently that release probabilities of readily releasable vesicles are heterogeneous. Here, we show that cAMP selectively increases the number of vesicles with higher release probabilities, whereas a slow component of the EPSC, representing vesicles that fuse more slowly, is unchanged. cAMP increases the apparent Ca2+ sensitivity for secretion, but this increase does not reflect an increase in release probability necessarily but rather an increase in the number of highly sensitive vesicles.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Potentiation of transmitter release by protein kinase C in goldfish retinal bipolar cells.

1. We examined whether transmitter release could be modified by the activation of protein kinase C (PKC) of retinal bipolar cells. A bipolar cell with a large axon terminal was isolated from the goldfish retina. The presynaptic Ca2+ current was measured under whole-cell voltage clamp, and the released transmitter (probably glutamate) was detected electrophysiologically by using the response of NMDA receptors of catfish horizontal cells as a reporter. 2. Transmitter release was potentiated by a PKC activator, phorbol 12-myristate 13-acetate (PMA), but not by an ineffective phorbol ester, 4alpha-phorbol 12,13-didecanoate. A PKC inhibitor, bisindolylmaleimide I, did not affect the transmitter release by itself but blocked the PMA-induced potentiation of transmitter release. These results suggest that the actions of PMA were mediated via the activation of PKC. 3. Introduction of 5 mM EGTA into the presynaptic terminals of bipolar cells revealed two separate components of transmitter release. A rapid component was triggered immediately after depolarization while a slow component appeared with a delay. Application of PMA selectively potentiated the slow component without affecting the Ca2+ dependence of exocytosis. 4. We suggest that the activation of PKC may modify the recruitment process of synaptic vesicles in retinal bipolar cells.

Animals↗

PubMed1998

Submillisecond kinetics of glutamate release from a sensory synapse.

Exocytosis-mediated glutamate release from ribbon-type synaptic terminals of retinal bipolar cells was studied using AMPA receptors and simultaneous membrane capacitance measurements. Release onset (delay <0.8 ms) and offset were closely tied to Ca2+ channel opening and closing. Asynchronous release was not copious and we estimate that there are approximately 5 Ca2+ channels per docked synaptic vesicle. Depending on Ca2+ current amplitude, release occurred in a single fast bout or in two successive bouts with fast and slow onset kinetics. The second, slower bout may reflect a mobilization rate of reserve vesicles toward fusion sites that is accelerated by increasing Ca2+ influx. Bipolar cell synaptic ribbons thus are remarkably versatile signal transducers, capable of transmitting rapidly changing sensory input, as well as sustained stimuli, due to their large pool of releasable vesicles.

Animals↗

PubMed1997

Ca2+ -activated K+ current at presynaptic terminals of goldfish retinal bipolar cells.

Properties of the Ca2+ -activated K+ current (I[K(Ca)]) were investigated in bipolar cells isolated from the goldfish retina. Pharmacological experiments and single channel current recordings demonstrated that I[K(Ca)] represented currents through BK channels, which were confined mostly to the presynaptic terminal. The ensemble noise analysis of I[K(Ca)], which was evoked following the activation of presynaptic Ca2+ current, revealed that the single channel conductance and open probability (P(o)) were approximately 50 pS ([K+]o = 2.6 mM, [K+]i = 140 mM) and 0.6 at 0 mV, respectively. To estimate [Ca2+]i at the cytosolic side of BK channels, activation of I[K(Ca)] was examined in Ca2+-loaded bipolar cells bathed in Co2+ solution. [Ca2+]i was monitored using furaptra fluorimetry. It was found that [Ca2+]i ranged between 10 and 20 microM when P(o) was 0.6. A high concentration of BAPTA ( > 20 mM) was required to suppress I[K(Ca)]. Under this condition, channel number was reduced without changing P(o). Therefore, it is likely that some BK channels are co-localized with Ca2+ channels in presynaptic terminals of retinal bipolar cells.

Animals↗

PubMed1997

Two components of transmitter release in retinal bipolar cells: exocytosis and mobilization of synaptic vesicles.

Ca2+-transmitter release coupling was examined using bipolar cells with large presynaptic terminals dissociated from the goldfish retina. Presynaptic Ca2+ current (I(Ca)) was recorded under the whole-cell voltage clamp. Release of excitatory amino acid transmitter was simultaneously monitored as the current through N-methyl-D-asperate (NMDA) receptors of reporter cells or as the membrane capacitance (C(m)) change associated with exocytosis. When I(Ca) was activated by a long depolarizing pulse, a double-peaked transmitter-induced current (I(tr)) was elicited in reporter cells. The rapid component of I(tr) was evoked immediately after the onset of depolarization, and was affected only slightly by intracellularly applied Ca2+ chelators. The delayed slow component of I(tr) was elicited during depolarization once a fixed amount of Ca2+ was accumulated in presynaptic terminals, and its appearance was suppressed or retarded by Ca2+ chelators. Two components of transmitter release were also recognized by monitoring C(m) changes elicited by the activation of I(Ca). These results suggest that bipolar cells have at least two pools of synaptic vesicles; a small, immediately releasable pool and a large releasable pool. The rapid and the delayed slow components of transmitter release may reflect exocytosis and mobilization of synaptic vesicles, respectively.

Animals↗

PubMed1995

Potentiation of Ca2+ transients in the presynaptic terminals of goldfish retinal bipolar cells.

1. To study a possible contribution of intracellular Ca2+ stores to the presynaptic Ca2+ regulation, the Ca2+ current (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were simultaneously monitored in isolated goldfish retinal bipolar cells using the whole-cell voltage clamp procedure and fura-2 fluorimetry. 2. The Ca2+ transient triggered by the activation of ICa was potentiated when [Ca2+]i was increased by applying either a prepulse or a small steady depolarization. The potentiation seemed to be partly due to the release of Ca2+ from intracellular Ca2+ stores. 3. The intracellular Ca2+ release was reversibly inhibited by caffeine but was not affected by ryanodine, suggesting that Ca2+ is released through intracellular Ca2+ channels which differ from ryanodine receptor channels. 4. These results suggest that the intracellular Ca2+ release may contribute to the facilitation of transmitter release.

Animals↗