Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held.

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

Heterogeneous Ca2+ influx along the adult calyx of Held: a structural and computational study.

The calyx of Held is a morphologically complex nerve terminal containing hundreds to thousands of active zones. The calyx must support high rates of transient, sound-evoked vesicular release superimposed on a background of sustained release, due to the high spontaneous rates of some afferent fibers. One means of distributing vesicle release in space and time is to have heterogeneous release probabilities (Pr) at distinct active zones, which has been observed at several CNS synapses including the calyx of Held. Pr may be modulated by vesicle proximity to Ca2+ channels, by Ca2+ buffers, by changes in phosphorylation state of proteins involved in the release process, or by local variations in Ca2+ influx. In this study, we explore the idea that the complex geometry of the calyx also contributes to heterogeneous Pr by impeding equal propagation of action potentials through all calyx compartments. Given the difficulty of probing ion channel distribution and recording from adult calyces, we undertook a structural and modeling approach based on computerized reconstructions of calyces labeled in adult cats. We were thus able to manipulate placement of conductances and test their effects on Ca2+ concentration in all regions of the calyx following an evoked action potential in the calyceal axon. Our results indicate that with a non-uniform distribution of Na+ and K+ channels, action potentials do not propagate uniformly into the calyx, Ca2+ influx varies across different release sites, and latency for these events varies among calyx compartments. We suggest that the electrotonic structure of the calyx of Held, which our modeling efforts indicate is very sensitive to the axial resistivity of cytoplasm, may contribute to variations in release probability within the calyx.

Structure suggests function: the case for synaptic ribbons as exocytotic nanomachines.

Synaptic ribbons, the organelles identified in electron micrographs of the sensory synapses involved in vision, hearing, and balance, have long been hypothesized to play an important role in regulating presynaptic function because they associate with synaptic vesicles at the active zone. Their physiology and molecular composition have, however, remained largely unknown. Recently, a series of elegant studies spurred by technical innovation have finally begun to shed light on the ultrastructure and function of ribbon synapses. Electrical capacitance measurements have provided sub-millisecond resolution of exocytosis, evanescent-wave microscopy has filmed the fusion of single 30 nm synaptic vesicles, electron tomography has revealed the 3D architecture of the synapse, and molecular cloning has begun to identify the proteins that make up ribbons. These results are consistent with the ribbon serving as a vesicle "conveyor belt" to resupply the active zone, and with the suggestion that ribbon and conventional chemical synapses have much in common.

Light evokes Ca2+ spikes in the axon terminal of a retinal bipolar cell.

Bipolar cells in the vertebrate retina have been characterized as nonspiking interneurons. Using patch-clamp recordings from goldfish retinal slices, we find, however, that the morphologically well-defined Mb1 bipolar cell is capable of generating spikes. Surprisingly, in dark-adapted retina, spikes were reliably evoked by light flashes and had a long (1-2 s) refractory period. In light-adapted retina, most Mb1 cells did not spike. However, an L-type Ca2+ channel agonist could induce periodic spiking in these cells. Spikes were determined to be Ca2+ action potentials triggered at the axon terminal and were abolished by 2-amino-4-phosphonobutyric acid (APB), an agonist that mimics glutamate. Signaling via spikes in a specific class of bipolar cells may serve to accelerate and amplify small photo-receptor signals, thereby securing the synaptic transmission of dim and rapidly changing visual input.

Fine-tuning an auditory synapse for speed and fidelity: developmental changes in presynaptic waveform, EPSC kinetics, and synaptic plasticity.

Fast, precise, and sustained synaptic transmission at high frequency is thought to be crucial for the task of sound localization in the auditory brainstem. However, recordings from the calyx of Held synapse have revealed severe frequency-dependent synaptic depression, which tends to degrade the exact timing of postsynaptic spikes. Here we investigate the functional changes occurring throughout the critical period of synapse refinement from immature calyx terminal [postnatal day 5 (P5)] to after the onset of hearing (P12-P14). Surprisingly, for recordings near physiological temperature (35 degrees C), we find that P14 synapses are already able to follow extremely high input rates of up to 800 Hz. This ability stems in part from a remarkable shortening of presynaptic action potentials, which may lead to a lowering of release probability and decrease in synaptic delays during development. In addition, AMPA receptor-mediated EPSCs as well as quantal synaptic currents acquired progressively faster kinetics, although their mean amplitudes did not change significantly. NMDA receptor-mediated EPSCs, however, diminished with age, as indicated by a 50% reduction in mean amplitude and faster decay kinetics. Finally, the degree of synaptic depression was greatly attenuated with age, presumably because of a 2.5-fold or larger increase in the releasable pool of vesicles, which together with a decreasing release probability produces a fairly constant EPSC amplitude. This finely tuned orchestra of developmental changes thus simultaneously promotes speed while preventing premature vesicle pool depletion during prolonged bouts of firing. A few critical days in postnatal development can thus have a large impact on synaptic function.

Electrophysiology of synaptic vesicle cycling.

Patch-clamp capacitance measurements can monitor in real time the kinetics of exocytosis and endocytosis in living cells. We review the application of this technique to the giant presynaptic terminals of goldfish bipolar cells. These terminals secrete glutamate via the fusion of small, clear-core vesicles at specialized, active zones of release called synaptic ribbons. We compare the functional characteristics of transmitter release at ribbon-type and conventional synapses, both of which have a unique capacity for fast and focal vesicle fusion. Subsequent rapid retrieval and recycling of fused synaptic vesicle membrane allow presynaptic terminals to function independently of the cell soma and, thus, as autonomous computational units. Together with the mobilization of reserve vesicle pools, local cycling of synaptic vesicles may delay the onset of vesicle pool depletion and sustain neuronal output during high stimulation frequencies.

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.

Presynaptic depression at a calyx synapse: the small contribution of metabotropic glutamate receptors.

Synaptic depression of evoked EPSCs was quantified with stimulation frequencies ranging from 0.2 to 100 Hz at the single CNS synapse formed by the calyx of Held in the rat brainstem. Half-maximal depression occurred at approximately 1 Hz, with 10 and 100 Hz stimulation frequencies reducing EPSC amplitudes to approximately 30% and approximately 10% of their initial magnitude, respectively. The time constant of recovery from depression elicited by 10 Hz afferent fiber stimulation was 4.2 sec. AMPA and NMDA receptor-mediated EPSCs depressed in parallel at 1-5 Hz stimulation frequencies, suggesting that depression was induced by presynaptic mechanism(s) that reduced glutamate release. To determine the contribution of autoreceptors to depression, we studied the inhibitory effects of the metabotropic glutamate receptor (mGluR) agonists (1S, 3S)-ACPD and L-AP4 and found them to be reversed in a dose-dependent manner by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), a novel and potent competitive antagonist of mGluRs. At 300 microM, CPPG completely reversed the effects of L-AP4 and (1S, 3S)-ACPD, but reduced 5-10 Hz elicited depression by only approximately 6%. CPPG-sensitive mGluRs, presumably activated by glutamate spillover during physiological synaptic transmission, thus contribute on the order of only 10% to short-term synaptic depression. We therefore suggest that the main mechanism contributing to the robust depression elicited by 5-10 Hz afferent fiber stimulation of the calyx of Held synapse is synaptic vesicle pool depletion.

Depletion and replenishment of vesicle pools at a ribbon-type synaptic terminal.

Synaptic depression was studied using capacitance measurements in synaptic terminals of retinal bipolar neurons. Single 250 msec depolarizations evoked saturating capacitance responses averaging approximately 150 fF, whereas trains of 250 msec depolarizations produced plateau capacitance increases of approximately 300 fF. Both types of stimuli were followed by pronounced synaptic depression, which recovered with a time constant of approximately 8 sec after single pulses but required >20 sec for full recovery after pulse trains. Inactivation of presynaptic calcium current could not account for depression, which is attributed instead to depletion of releasable and reserve vesicle pools that are recruited and replenished at different rates. Recovery from depression was normal in the absence of fast endocytosis, suggesting that replenishment was from a reserve pool of preformed vesicles rather than from preferential recycling of recently fused vesicles. Given the in vivo light response of the class of bipolar neuron studied here, it is likely that, under at least some illumination conditions, the cells produce a fast and phasic bout of exocytosis rather than tonic release.

Evidence that vesicles on the synaptic ribbon of retinal bipolar neurons can be rapidly released.

We relate the ultrastructure of the giant bipolar synapse in goldfish retina to the jump in capacitance that accompanies depolarization-evoked exocytosis. Mean vesicle diameter is 29 +/- 4 nm, giving 26.4 aF/vesicle, so the maximum evoked capacitance (150 fF within 200 ms) represents fusion of about 5700 vesicles. Two terminals contained, respectively, 45 and 65 ribbon-type synaptic outputs, and a fully loaded ribbon tethers about 110 vesicles. Thus, the tethered pool, about 6000 vesicles, corresponds to the rapidly released pool. Further, the difference between small and large terminals in number of tethered vesicles matches their difference in capacitance jump. This suggests, within a "fire and reload" model of exocytosis, that the ribbon translocates synaptic vesicles very rapidly to membrane docking sites, supporting a maximum release rate of 500 vesicles/active zone/s, until the population of tethered vesicles is exhausted.

Calcium-dependent inactivation of calcium current in synaptic terminals of retinal bipolar neurons.

Giant synaptic terminals (approximately 10 micrometer diameter) of bipolar neurons from goldfish retina were used to directly investigate calcium-dependent inactivation of presynaptic calcium current. During sustained depolarization, calcium current was initially constant for a period lasting up to several hundred milliseconds and then it declined exponentially. The duration of the initial delay was shorter and the rate of inactivation was faster with larger calcium current. The fastest time constant of inactivation (in the range of 2-5 sec) was observed under weak calcium buffering conditions. Inactivation was attenuated when external Ca2+ was replaced with Ba2+ and when terminals were dialyzed with high concentrations of internal BAPTA. Elevation of intracellular calcium concentration ([Ca2+]i) by application of the calcium ionophore ionomycin or by dialysis with pipette solutions containing buffered elevated [Ca2+] produced inactivation of calcium current. The rate of recovery from inactivation was not determined by the recovery of [Ca2+]i to baseline after a stimulus. The results demonstrate that the presynaptic calcium current in bipolar neurons is inactivated by elevated [Ca2+]i, but the inactivation is approximately 100-fold slower than previously described calcium-dependent inactivation in other types of cells.

Inhibition of endocytosis by elevated internal calcium in a synaptic terminal.

During synaptic transmission in the nervous system, synaptic vesicles fuse with the plasma membrane of presynaptic terminals, releasing neurotransmitter by exocytosis. The vesicle membrane is then retrieved by endocytosis and recycled into new transmitter-containing vesicles. Exocytosis in synaptic terminals is calcium-dependent, and we now report that endocytosis also is regulated by the intracellular calcium concentration ([Ca2+]i). Capacitance measurements in synaptic terminals of retinal bipolar neurons revealed that endocytosis was strongly inhibited by elevated [Ca2+]i in the range achieved by Ca(2+)-current activation. The rate of membrane retrieval was steeply dependent on [Ca2+]i, with a Hill coefficient of 4 and half-inhibition at approximately 500 nM. At [Ca2+]i > or = 900 nM, endocytosis was entirely absent. The action of internal calcium on endocytosis represents a novel negative-feedback mechanism controlling the rate of membrane recovery in synaptic terminals after neurotransmitter secretion. As membrane retrieval is the first step in vesicle recycling, this mechanism may contribute to activity-dependent synaptic depression.

Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals.

Communication among neurons occurs at specialized synaptic junctions, where neurotransmitter is released via calcium-dependent exocytosis from the synaptic terminal of the presynaptic cell onto the postsynaptic target neuron. Here we exploit the unique properties of giant synaptic terminals of bipolar neurons from goldfish retina to establish the kinetics and calcium-dependence of exocytosis, and the characteristics of membrane retrieval following secretion in presynaptic terminals. Simultaneous patch-clamp, calcium-indicator dye and time-resolved capacitance measurements reveal that activation of calcium current drives secretion at a rapid rate of about 10,000 vesicles per s and the calcium level necessary to drive secretion is locally greater than 50 microM. Two components of membrane retrieval were observed following secretory stimulation. After strong stimulation, capacitance returned to rest with a time constant of about 30 s, but after weaker stimuli recovery was much faster, with a time constant of about 2 s. Secretion in a vertebrate central nervous system neuron was thus found to differ substantially from that in other secretory cells in its rapid rate of vesicle fusion, requirement for high levels of intracellular calcium, and the high speed and completeness of membrane retrieval. These distinctive features reflect the specialization of neuronal synaptic terminals for rapid and focally directed release of neurotransmitter.