Henrique von Gersdorff

Short-term plasticity at the calyx of held

Synapses show widely varying degrees of short-term facilitation and depression. Several mechanisms have been proposed to underlie short-term plasticity, but the contributions of presynaptic mechanisms have been particularly difficult to study because of the small size of synaptic boutons in the mammalian brain. Here we review the functional properties of the calyx of Held, a giant nerve terminal that has shed new light on the general mechanisms that control short-term plasticity. The calyx of Held has also provided fresh insights into the strategies used by synapses to extend their dynamic range of operation and preserve the timing of sensory stimuli.

Optimizing Synaptic Architecture and Efficiency for High-Frequency Transmission

Bursts of neuronal activity are transmitted more effectively as synapses mature. However, the mechanisms that control synaptic efficiency during development are poorly understood. Here, we study postnatal changes in synaptic ultrastructure and exocytosis in a calyx-type nerve terminal. Vesicle pool size, exocytotic efficiency (amount of exocytosis per Ca influx), Ca current facilitation, and the number of active zones (AZs) increased with age, whereas AZ area, number of docked vesicles per AZ, and release probability decreased with age. These changes led to AZs that are less prone to multivesicular release, resulting in reduced AMPA receptor saturation and desensitization. A greater multiplicity of small AZs with few docked vesicles, a larger pool of releasable vesicles, and a higher efficiency of release thus promote prolonged high-frequency firing in mature synapses.

Physiological Temperatures Reduce the Rate of Vesicle Pool Depletion and Short-Term Depression via an Acceleration of Vesicle Recruitment

The timing and strength of synaptic transmission is profoundly dependent on temperature. However, the temperature dependence of the multiple mechanisms that contribute to short-term synaptic plasticity is poorly understood. Here, we use voltage-clamp recordings to quantify the temperature dependence of exocytosis at the calyx of Held synapse. EPSC and miniature EPSC amplitudes were larger at physiological temperature, but quantal content during low-frequency (0.05 Hz) stimulation was constant after temperature jumps from 22–24°C to 35–37°C. The initial degree of EPSC depression during 100 Hz stimuli trains was unchanged with temperature, as were estimates of release probability and vesicle pool size. In contrast, physiological temperatures dramatically relieved depression measured after 40 stimuli at 100 Hz by increasing twofold the rate of recovery from depression. Presynaptic calyx recordings revealed that physiological temperature increased capacitance jumps resulting from 0.5 and 1 ms depolarizations by increasing Ca2+ influx. When Ca2+ entry was equalized at the two temperatures, exocytosis exhibited little temperature dependence for brief depolarizations. However, in response to longer depolarizations, raising temperature increased a slow phase of exocytosis, without affecting overall Ca2+ entry or the size of the readily releasable pool of vesicles. Higher temperatures also increased the rate of presynaptic Ca2+ current inactivation; nevertheless, the degree of steady-state EPSC depression was greatly reduced. Our results thus suggest that changes in steady-state EPSCs during stimulus trains at physiological temperature reflect larger quantal amplitudes and faster refilling of synaptic vesicle pools, leading to reduced short-term depression during prolonged high-frequency firing.

The Unitary Event Underlying Multiquantal EPSCs at a Hair Cell's Ribbon Synapse

EPSCs at the synapses of sensory receptors and of some CNS neurons include large events thought to represent the synchronous release of the neurotransmitter contained in several synaptic vesicles by a process known as multiquantal release. However, determination of the unitary, quantal size underlying such putatively multiquantal events has proven difficult at hair cell synapses, hindering confirmation that large EPSCs are in fact multiquantal. Here, we address this issue by performing presynaptic membrane capacitance measurements together with paired recordings at the ribbon synapses of adult hair cells. These simultaneous presynaptic and postsynaptic assays of exocytosis, together with electron microscopic estimates of single vesicle capacitance, allow us to estimate a single vesicle EPSC charge of approximately −45 fC, a value in close agreement with the mean postsynaptic charge transfer of uniformly small EPSCs recorded during periods of presynaptic hyperpolarization. By thus establishing the magnitude of the fundamental quantal event at this peripheral sensory synapse, we provide evidence that the majority of spontaneous and evoked EPSCs are multiquantal. Furthermore, we show that the prevalence of uniquantal versus multiquantal events is Ca2+ dependent. Paired recordings also reveal a tight correlation between membrane capacitance increase and evoked EPSC charge, indicating that glutamate release during prolonged hair cell depolarization does not significantly saturate or desensitize postsynaptic AMPA receptors. We propose that the large EPSCs reflect the highly synchronized release of multiple vesicles at single presynaptic ribbon-type active zones through a compound or coordinated vesicle fusion mechanism.

Presynaptic Na+ Channels: Locus, Development, and Recovery from Inactivation at a High-Fidelity Synapse

Na+ channel recovery from inactivation limits the maximal rate of neuronal firing. However, the properties of presynaptic Na+ channels are not well established because of the small size of most CNS boutons. Here we study the Na+ currents of the rat calyx of Held terminal and compare them with those of postsynaptic cells. We find that presynaptic Na+ currents recover from inactivation with a fast, single-exponential time constant (24°C, τ of 1.4-1.8 ms; 35°C, τ of 0.5 ms), and their inactivation rate accelerates twofold during development, which may contribute to the shortening of the action potential as the terminal matures. In contrast, recordings from postsynaptic cells in brainstem slices, and acutely dissociated, reveal that their Na+ currents recover from inactivation with a double-exponential time course (τfast of 1.2-1.6 ms; τslow of 80-125 ms; 24°C). Surprisingly, confocal immunofluorescence revealed that Na+ channels are mostly absent from the calyx terminal but are instead highly concentrated in an unusually long (≈20-40 μm) unmyelinated axonal heminode. Outside-out patch recordings confirmed this segregation. Expression of Nav1.6 α-subunit increased during development, whereas the Nav1.2α-subunit was not present. Serial EM reconstructions also revealed a long pre-calyx heminode, and biophysical modeling showed that exclusion of Na+ channels from the calyx terminal produces an action potential waveform with a shorter half-width. We propose that the high density and polarized locus of Na+ channels on a long heminode are critical design features that allow the mature calyx of Held terminal to fire reliably at frequencies near 1 kHz.

Sharp Ca2+ Nanodomains beneath the Ribbon Promote Highly Synchronous Multivesicular Release at Hair Cell Synapses

Hair cell ribbon synapses exhibit several distinguishing features. Structurally, a dense body, or ribbon, is anchored to the presynaptic membrane and tethers synaptic vesicles; functionally, neurotransmitter release is dominated by large EPSC events produced by seemingly synchronous multivesicular release. However, the specific role of the synaptic ribbon in promoting this form of release remains elusive. Using complete ultrastructural reconstructions and capacitance measurements of bullfrog amphibian papilla hair cells dialyzed with high concentrations of a slow Ca2+ buffer (10 mm EGTA), we found that the number of synaptic vesicles at the base of the ribbon correlated closely to those vesicles that released most rapidly and efficiently, while the rest of the ribbon-tethered vesicles correlated to a second, slower pool of vesicles. Combined with the persistence of multivesicular release in extreme Ca2+ buffering conditions (10 mm BAPTA), our data argue against the Ca2+-dependent compound fusion of ribbon-tethered vesicles at hair cell synapses. Moreover, during hair cell depolarization, our results suggest that elevated Ca2+ levels enhance vesicle pool replenishment rates. Finally, using Ca2+ diffusion simulations, we propose that the ribbon and its vesicles define a small cytoplasmic volume where Ca2+ buffer is saturated, despite 10 mm BAPTA conditions. This local buffer saturation permits fast and large Ca2+ rises near release sites beneath the synaptic ribbon that can trigger multiquantal EPSCs. We conclude that, by restricting the available presynaptic volume, the ribbon may be creating conditions for the synchronous release of a small cohort of docked vesicles.

Synaptic Activation of Presynaptic Glutamate Transporter Currents in Nerve Terminals

Glutamate uptake by high-affinity transporters is responsible for limiting the activation of postsynaptic receptors and maintaining low levels of ambient glutamate. The reuptake process generates membrane currents, which can be activated by synaptically released glutamate in glial cells and some postsynaptic neurons. However, less is known about presynaptic transporter currents because the small size of synaptic boutons precludes direct recordings. Here, we have recorded from two giant nerve terminals: bipolar cell synaptic terminals in goldfish retina and the calyx of Held in rat auditory brainstem. Exocytosis was evoked by brief depolarizations and measured as an increase in membrane capacitance. In isolated bipolar cell terminals, exocytosis was associated with an anion (NO3- or Cl-) current. The current peaked 2.8 msec after the start of the depolarization and decayed with a mean time constant of 8.5 msec. It was inhibited by the nontransportable glutamate transporter antagonist sc-threo-β-benzyloxyaspartate (TBOA) but was insensitive to the GLT1/EAAT2 subtype-selective antagonist dihydrokainate and was affected by extracellular pH buffering. A TBOA-sensitive anion current was also evoked by application of exogenous glutamate to bipolar cell terminals. The large single-channel conductance, derived from noise analysis, and previous immunolocalization studies suggest that synaptically released glutamate activates EAAT5-type transporters in bipolar cell terminals. In contrast, neither exocytosis nor exogenous glutamate evoked a transporter current in the calyx of Held. Glutamate transporter currents with rapid kinetics are therefore identified and characterized in bipolar cell terminals, providing a valuable system for investigating the function and modulation of presynaptic glutamate transporters.

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.

Retroinhibition of Presynaptic Ca2+ Currents by Endocannabinoids Released via Postsynaptic mGluR Activation at a Calyx Synapse

We investigated the mechanisms by which activation of group I metabotropic glutamate receptors (mGluRs) and CB1 cannabinoid receptors (CB1Rs) leads to inhibition of synaptic currents at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of the rat auditory brainstem. In ∼50% of the MNTB neurons tested, activation of group I mGluRs by the specific agonist (s)-3,5-dihydroxyphenylglycine (DHPG) reversibly inhibited AMPA receptor- and NMDA receptor-mediated EPSCs to a similar extent and reduced paired-pulse depression, suggestive of an inhibition of glutamate release. Presynaptic voltage-clamp experiments revealed a reversible reduction of Ca2+ currents by DHPG, with no significant modification of the presynaptic action potential waveform. Likewise, in ∼50% of the tested cells, the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN) reversibly inhibited EPSCs, presynaptic Ca2+ currents, and exocytosis. For a given cell, the amount of inhibition by DHPG correlated with that by WIN. Moreover, the inhibitory action of DHPG was blocked by the CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and occluded by WIN, indicating that DHPG and WIN operate via a common pathway. The inhibition of EPSCs by DHPG, but not by WIN, was abolished after dialyzing 40 mm BAPTA into the postsynaptic cell, suggesting that DHPG activated postsynaptic mGluRs. Light and electron microscopy immunolabeling indicated a presynaptic expression of CB1Rs and postsynaptic localization of mGluR1a. Our data suggest that activation of postsynaptic mGluRs triggers the Ca2+-dependent release of endocannabinoids that activate CB1 receptors on the calyx terminal, which leads to a reduction of presynaptic Ca2+ current and glutamate release.

Presynaptic Ca2+ buffers control the strength of a fast post-tetanic hyperpolarization mediated by the α3 Na+/K+-ATPase

The excitability of CNS presynaptic terminals after a tetanic burst of action potentials is important for synaptic plasticity. The mechanisms that regulate excitability, however, are not well understood. Using direct recordings from the rat calyx of Held terminal, we found that a fast Na+/K+-ATPase (NKA)-mediated post-tetanic hyperpolarization (PTH) controls the probability and precision of subsequent firing. Notably, increasing the concentration of internal Ca2+ buffers or decreasing Ca2+ influx led to larger PTH amplitudes, indicating that an increase in [Ca2+]i regulates PTH via inhibition of NKAs. The characterization for the first time of a presynaptic NKA pump current, combined with immunofluorescence staining, identified the α3-NKA isoform on calyx terminals. Accordingly, the increased ability of the calyx to faithfully fire during a high-frequency train as it matures is paralleled by a larger expression of α3-NKA during development. We propose that this newly discovered Ca2+ dependence of PTH is important in the post-burst excitability of nerve terminals.