Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

RIM determines Ca²+ channel density and vesicle docking at the presynaptic active zone

At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles. The mechanisms that enrich Ca2+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knockout approach at a presynaptically accessible central nervous system synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca2+ channel density, revealing a role of RIM proteins in Ca2+ channel targeting. Removal of RIMs also reduced the readily releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca2+ channel-vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release, enabling a high presynaptic Ca2+ channel density and vesicle docking at the active zone.     

Properties of a model of Ca(++)-dependent vesicle pool dynamics and short term synaptic depression

We explore the properties of models of synaptic vesicle dynamics, in which synaptic depression is attributed to depletion of a pool of release-ready vesicles. Two alternative formulations of the model allow for either recruitment of vesicles from an unlimited reserve pool (vesicle state model) or for recovery of a fixed number of release sites to a release-ready state (release-site model). It is assumed that, following transmitter release, the recovery of the release-ready pool of vesicles is regulated by the intracellular free Ca(++) concentration, [Ca(++)](i). Considering the kinetics of [Ca(++)](i) after single presynaptic action potentials, we show that pool recovery can be described by two distinct kinetic components. With such a model, complex kinetic and steady-state properties of synaptic depression as found in several types of synapses can be accurately described. However, the specific assumption that enhanced recovery is proportional to [Ca(++)](i), as measured with Ca(++) indicator dyes, is not confirmed by experiments at the calyx of Held, in which [Ca(++)](i)-homeostasis was altered by adding low concentrations of the exogenous Ca(++) buffer, fura-2, to the presynaptic terminal. We conclude that synaptic depression at the calyx of Held is governed by localized, near membrane [Ca(++)](i) signals not visible to the indicator dye, or else by an altogether different mechanism. We demonstrate that, in models in which a Ca(++)-dependent process is linearly related to [Ca(++)](i), the addition of buffers has only transient but not steady-state consequences.     

Augmentation is a potentiation of the exocytotic process

Short-term synaptic enhancement is caused by an increase in the probability with which synaptic terminals release transmitter in response to presynaptic action potentials. Since exocytosed vesicles are drawn from a readily releasable pool of packaged transmitter, enhancement must result either from an increase in the size of the pool or an elevation in the fraction of releasable vesicles that undergoes exocytosis with each action potential. We show here that at least one major component of enhancement, augmentation, is not caused by an increase in the size of the readily releasable pool but is instead associated with an increase in the efficiency with which action potentials induce the exocytosis of readily releasable vesicles.     

Heterogeneity of the Ca2+ sensitivity of secretion in a pituitary gonadotrope cell line and its modulation by protein kinase C and Ca2+

Modulation of the Ca2+ sensitivity and cooperativity of secretion is an important means of regulating neurotransmission and hormone secretion. Employing high-time resolution measurement of membrane capacitance (Cm) stimulated by step-like or ramp [Ca2+]i elevation, we have identified the co-existence of both a high and low Ca2+-sensitive exocytosis in an immortal pituitary gonadotrope cell line, LbetaT2. Ramp [Ca2+]i generated by slow uncaging elicited a biphasic C(m) response. The first phase of response, which represents a highly Ca2+-sensitive pool (HCSP) of vesicles, began to secrete at low [Ca2+]i concentration (<1 microM) with low Ca2+ cooperativity. In contrast, the second phase, which represents a lowly Ca2+-sensitive pool (LCSP) of vesicles, only exocytozed at higher [Ca2+]i (>5 microM) and displayed a steep Ca2+ cooperativity. The co-existence of vesicle populations with different Ca2+ sensitivities was further confirmed by flash photolysis stimuli. The size of the HCSP was approximately 30 fF under resting conditions, but was dramatically increased (approximately threefold) by application of phorbol-12-myristate-13-acetate (PMA, an activator of protein kinase C). Forskolin (an activator of protein kinase A), however, exerted no significant effect on the size of both HCSP and LCSP. GnRH (gonadotropin releasing hormone) augmented the size of both pools to a larger extent (5- and 1.7-fold increase for HCSP and LCSP, respectively). The heterogeneity of Ca2+ sensitivity from different pools of vesicles and its differential modulation by intracellular signals suggests that LbetaT2 cells are an ideal model to further unravel the mechanism underlying the modulation of Ca2+-sensing machineries for exocytosis.     

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.     

应用电镜X射线显微分析方法研究神经细胞内钙离子的分布

本文应用X射线能谱分析结合电镜技术研究了钙离子在青蛙交感神经节神经元内的分布及其在茶碱作用下分布的变化.实验结果表明在组织样品的电子致密沉积物EDD中含有钙离子成分.在青蛙交感神经节突触后神经元中,包含钙离子的EDD存在于质膜、亚表面池及线粒体中;在突触前神经末梢中,突触小泡的膜上也可观察到EDD.在茶碱作用下,交感神经节神经元的质膜、线粒体中的EDD大大地减少;在亚表面池中则没有或很少观察到EDD;突触前末梢中的突触小泡明显地趋向聚集,在突触小泡之间的连接处频繁地出现EDD.本文根据实验结果讨论了茶碱可能促使钙离子从交感神经元的上述部位中释放出来,并认为质膜、亚表面池和线粒体是细胞内钙离子的贮存部位,而亚表面池可能是主要的贮存释放部位.突触前神经末梢内形态上的变化可能与神经递质释放的机理有关.     

Effects of adenosine on Ca2+ entry in the nerve terminal of the frog neuromuscular junction

This study aimed to test whether nerve-evoked and adenosine-induced synaptic depression are due to reduction in Ca2+ entry in nerve terminals of the frog neuromuscular junction. Nerve terminals were loaded with the fluorescent Ca2+ indicator fluo 3 (fluo 3-AM) or loaded with dextran-coupled Ca2+ green-1 transported from the cut end of the nerve. Adenosine (10-50 microM) did not change the resting level of Ca2+ in the presynaptic terminal, whereas it induced large Ca2+ responses in perisynaptic Schwann cells, indicating that adenosine was active and might have induced changes in the level of Ca2+ in the nerve terminal. Ca2+ responses in nerve terminals could be induced by nerve stimulation (0.5 or 100 Hz for 100 ms) over several hours. In the presence of adenosine (10 microM), the size and duration of the nerve-evoked Ca2+ responses were unchanged. When extracellular Ca2+ concentration was lowered to produce the same reduction in transmitter release as the application of adenosine, Ca2+ responses induced by nerve stimulations were reduced by 40%. This indicates that changes in Ca2+ responsible for the decrease in release should have been detected if the mechanism of adenosine depression involved partial block of Ca2+ influx. Ca2+ responses evoked by prolonged high frequency trains of stimuli (50 Hz for 10 or 30 s), which caused profound depression of transmitter release, were sustained during the whole duration of the stimulation, and adenosine had no effect on these responses. These data indicate that neither adenosine induced synaptic depression nor stimulation-induced synaptic depression are caused by reductions in Ca2+ entry into the presynaptic terminal in the frog neuromuscular junction.     

Modulation of transmitter release by presynaptic resting potential and background calcium levels

Activation of presynaptic ion channels alters the membrane potential of nerve terminals, leading to changes in transmitter release. To study the relationship between resting potential and exocytosis, we combined pre- and postsynaptic electrophysiological recordings with presynaptic Ca(2+) measurements at the calyx of Held. Depolarization of the membrane potential to between -60 mV and -65 mV elicited P/Q-type Ca(2+) currents of < 1 pA and increased intraterminal Ca(2+) by < 100 nM. These small Ca(2+) elevations were sufficient to enhance the probability of transmitter release up to 2-fold, with no effect on the readily releasable pool of vesicles. Moreover, the effects of mild depolarization on release had slow kinetics and were abolished by 1 mM intraterminal EGTA, suggesting that Ca(2+) acted through a high-affinity binding site. Together, these studies suggest that control of resting potential is a powerful means for regulating synaptic function at mammalian synapses.     

Calcium transients recorded with arsenazo III in the presynaptic terminal of the squid giant synapse

Transient changes in free intracellular Ca2+ concentration were monitored in the presynaptic terminal of the giant synapse of the squid, by means of the Ca2+-sensitive dye arsenazo III. Calibration experiments showed a linear relation between the amount of Ca2+ injected by iontophoresis into the terminal, and the peak size of the arsenazo light absorbance record. A light signal could be detected on tetanic stimulation of the presynaptic axon bathed in sea water containing 45 mM Ca2+. During a 1 s tetanus the light signal rose approximately linearly, even though transmitter release declined rapidly and the light signal subsequently declined with a half-time of 2-6 s. The Ca2+ transient elicited by single nerve impulses was recorded by signal averaging, and showed a time course very much slower than the duration of transmitter release.     

The coupling between synaptic vesicles and Ca2+ channels determines fast neurotransmitter release

In order to release neurotransmitter synchronously in response to a presynaptic action potential, synaptic vesicles must be both release competent and located close to presynaptic Ca2+ channels. It has not been shown, however, which of the two is the more decisive factor. We tested this issue at the calyx of Held synapse by combining Ca2+ uncaging and electrophysiological measurements of postsynaptic responses. After depletion of the synaptic vesicles that are responsible for synchronous release during action potentials, uniform elevation of intracellular Ca2+ by Ca2+ uncaging could still elicit rapid release. The Ca2+ sensitivity of remaining vesicles was reduced no more than 2-fold, which is insufficient to explain the slow-down of the kinetics of release (10-fold) observed during a depolarizing pulse. We conclude that recruitment of synaptic vesicles to sites where Ca2+ channels cluster, rather than fusion competence, is a limiting step for rapid neurotransmitter release in response to presynaptic action potentials.     

Mechanism of long-term potentiation of transmitter release induced by adrenaline in bullfrog sympathetic ganglia

A mechanism of the long-term potentiation of transmitter release induced by adrenaline (ALTP) was studied by recording intracellularly the fast excitatory postsynaptic potentials (fast EPSPs). The ALTP was produced during the blockade of K+ channels at the presynaptic terminals by tetraethylammonium (TEA). The synaptic delay, possibly reflecting a relative change in the duration of an action potential at the presynaptic terminal, was not changed during the course of the ALTP. By contrast, it was significantly lengthened by TEA and other K+ channel inhibitors (4-aminopyridine and Cs+) that markedly enhanced the evoked release of transmitter. The magnitude of facilitation of the fast EPSP, induced by a conditional stimulus to the preganglionic nerve, was decreased during the generation of the ALTP, but was unchanged during the potentiation of transmitter release caused by TEA. These results, together with theoretical considerations applying the residual Ca2+ hypothesis to the facilitation, suggest that the enhancement of transmitter release during the ALTP is not caused by an increased Ca2+ influx during a presynaptic impulse owing to the blockade of K+ channel or the modulation of Ca2+ channel, but presumably is induced by a rise in the basal level of free Ca2+ in the presynaptic terminal.     

Nanodomain coupling between Ca2+ channels and Ca2+ sensors promotes fast and efficient transmitter release at a cortical GABAergic synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse

Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i above 8 microM caused a biphasic capacitance increase corresponding to the fusion of approximately 40,000 vesicles. The kinetics of exocytosis displayed a fifth-order Ca2+ dependence reaching maximal rates of >3 x 10(7) vesicle/s. Exocytosis was always followed by slow, compensatory endocytosis (tau congruent with 15 s). Higher [Ca2+]i increased the contribution of a faster mode of endocytosis with a Ca2+ independent time constant of approximately 300 ms. These properties provide for rapid and sustained transmitter release from this large presynaptic terminal.     

Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices.

Many neurosecretory preparations display two components of depolarization-induced exocytosis: a phasic component synchronized with Ca2+ channel opening, followed by a slower sustained component. We evaluated possible mechanisms underlying this biphasic behavior by stimulating mouse chromaffin cells in situ with both depolarizations and flash photolysis of caged Ca2+. From a direct comparison of the secretory responses to both stimuli, we conclude that phasic and sustained release components originate from a readily releasable pool (RRP) of equally fusion-competent vesicles, suggesting that differences in the vesicles' proximity to Ca2+ channels underlie the biphasic secretory behavior. An intermediate pool in dynamic equilibrium with the RRP ensures rapid recruitment of release-ready vesicles after RRP depletion. Our results are discussed in terms of a refined model for secretion in chromaffin cells.     

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.     

Calcium signaling and synaptic modulation: regulation of endocannabinoid-mediated synaptic modulation by calcium

Postsynaptic Ca2+ signal influences synaptic transmission through multiple mechanisms. Some of them involve retrograde messengers that are released from postsynaptic neurons in a Ca2+-dependent manner and modulate transmitter release through activation of presynaptic receptors. Recent studies have revealed essential roles of endocannabinoids in retrograde modulation of synaptic transmission. Endocannabinoid release is induced by either postsynaptic Ca2+ elevation alone or activation of postsynaptic Gq/11-coupled receptors with or without Ca2+ elevation. The former pathway is independent of phospholipase Cbeta (PLCbeta) and requires a large Ca2+ elevation to a micromolar range. The latter pathway requires PLCbeta and is facilitated by a moderate Ca2+ elevation to a submicromolar range. This facilitation is caused by Ca2+-dependency of receptor-driven PLCbeta activation. The released endocannabinoids then activate presynaptic cannabinoid receptor type 1 (CB1), and suppress transmitter release from presynaptic terminals. Both CB1 receptors and Gq/11-coupled receptors are widely distributed in the brain. Thus, the endocannabinoid-mediated retrograde modulation may be an important and widespread mechanism in the brain, by which postsynaptic events including Gq/11-coupled receptor activation and Ca2+ elevation can retrogradely influence presynaptic function.     

Ca(2+) channels and transmitter release at the active zone

Ca(2+)-dependent transmitter release is the most important signaling mechanism for fast information transfer between neurons. Transmitter release takes places at highly specialized active zones with sub-micrometer dimension, which contain the molecular machinery for vesicle docking and -fusion, as well as a high density of voltage-gated Ca(2+) channels. In the absence of direct evidence for the ultrastructural localization of Ca(2+) channels at CNS synapses, important insights into Ca(2+) channel-vesicle coupling has come from functional experiments relating presynaptic Ca(2+) current and transmitter release, at large and accessible synapses like the calyx of Held. First, high slope values in log-log plots of transmitter release versus presynaptic Ca(2+) current indicate that multiple Ca(2+) channels are involved in release control of a single vesicle. Second, release kinetics in response to step-like depolarizations revealed fast- and slowly releasable sub-pools of vesicles, FRP and SRP, which, according to the "positional" model, are distinguished by a differential proximity to Ca(2+) channels. Considering recent evidence for a rapid conversion of SRP- to FRP vesicles, however, we highlight that multivesicular release events and clearance of vesicle membrane from the active zone must be taken into account when interpreting kinetic release data. We conclude that the careful kinetic analysis of transmitter release at presynaptically accessible and molecularly targeted synapses has the potential to yield important insights into the molecular physiology of transmitter release.     

Dynamic aspects of presynaptic calcium currents mediating synaptic transmission

Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers transmitter release. Direct recording of Ca2+ currents from the calyx of Held nerve terminal revealed that presynaptic VGCCs undergo various modulations via presynaptic G protein-coupled receptors (GPCRs), Ca2+-binding proteins and a developmental switch of their alpha1 subunits. Dynamic changes of presynaptic VGCCs alter synaptic efficacy, thereby contributing to a variety of modulations of the CNS function.