Geng n Li

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.

GABA Transporters Regulate a Standing GABAC Receptor-Mediated Current at a Retinal Presynaptic Terminal

At the axon terminal of goldfish retinal bipolar cells, GABAC receptors have been shown to mediate inhibitory reciprocal synaptic currents. Here, we demonstrate a novel standing GABAergic current mediated exclusively by GABAC receptors. Selective inhibition of GAT-1 GABA transporters on amacrine cells increases this tonic current and reveals a specific functional coupling between GAT-1 transporters and GABAC receptors. We propose that this GABAC receptor-mediated standing current serves to regulate synaptic gain by shunting depolarizing potentials that can produce Ca2+-dependent action potentials at the bipolar cell terminal. Furthermore, we find that the amount of GABAC receptor-mediated reciprocal feedback between bipolar cell terminals and amacrine cells is greatly increased when GAT-1 transporters are specifically blocked by NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride). The involvement of GAT-1 transporters in regulating this standing (or tonic) GABAC current implicates them in a novel role as major determinants of presynaptic excitability.

Recovery from Short-Term Depression and Facilitation Is Ultrafast and Ca2+ Dependent at Auditory Hair Cell Synapses

Short-term facilitation and depression coexist at many CNS synapses. Facilitation, however, has not been fully characterized at hair cell synapses. Using paired recordings and membrane capacitance measurements we find that paired-pulse plasticity at an adult frog auditory hair cell synapse depends on pulse duration and interpulse intervals. For short 20 ms depolarizing pulses, and interpulse intervals between 15 and 50 ms, facilitation occurred when hair cells were held at −90 mV. However, hair cells held at −60 mV displayed only paired-pulse depression. Facilitation was dependent on residual free Ca2+ levels because it was greatly reduced by the Ca2+ buffers EGTA and BAPTA. Furthermore, low external Ca2+ augmented facilitation, whereas depression was augmented by high external Ca2+, consistent with depletion of a small pool of fast releasing synaptic vesicles. Recovery from depression had a double-exponential time course with a fast component that may reflect the rapid replenishment of a depleted vesicle pool. We suggest that hair cells held at more depolarized in vivo-like resting membrane potentials have a tonic influx of Ca2+; they are thus in a dynamic state of continuous vesicle release, pool depletion and replenishment. Further Ca2+ influx during paired-pulse stimuli then leads to depression. However, at membrane potentials of −90 mV, ongoing release and pool depletion are minimized, so facilitation is revealed at time intervals when rapid vesicle pool replenishment occurs. Finally, we propose that vesicle pool replenishment kinetics is not rate limited by vesicle endocytosis, which is too slow to influence the rapid pool replenishment process.

Long-term plasticity mediated by mGluR1 at a retinal reciprocal synapse.

The flow of information across the retina is controlled by reciprocal synapses between bipolar cell terminals and amacrine cells. However, the synaptic delays and properties of plasticity at these synapses are not known. Here we report that glutamate release from goldfish Mb-type bipolar cell terminals can trigger fast (delay of 2-3 ms) and transient GABA(A) IPSCs and a much slower and more sustained GABA(C) feedback. Synaptically released glutamate activated mGluR1 receptors on amacrine cells and, depending on the strength of presynaptic activity, potentiated subsequent feedback. This poststimulus enhancement of GABAergic feedback lasted for up to 10 min. This form of mGluR1-mediated long-term synaptic plasticity may provide retinal reciprocal synapses with adaptive capabilities.

Single Ca2+ channels and exocytosis at sensory synapses.

Abstract  Hair cell synapses in the ear and photoreceptor synapses in the eye are the first synapses in the auditory and visual system. These specialized synapses transmit a large amount of sensory information in a fast and efficient manner. Moreover, both small and large signals with widely variable kinetics must be quickly encoded and reliably transmitted to allow an animal to rapidly monitor and react to its environment. Here we briefly review some aspects of these primary synapses, which are characterized by a synaptic ribbon in their active zones of transmitter release. We propose that these synapses are themselves highly specialized for the task at hand. Photoreceptor and bipolar cell ribbon synapses in the retina appear to have versatile properties that permit both tonic and phasic transmitter release. This allows them to transmit changes of both luminance and contrast within a visual field at different ambient light levels. By contrast, hair cell ribbon synapses are specialized for a highly synchronous form of multivesicular release that may be critical for phase locking to low‐frequency sound‐evoked signals at both low and high sound intensities. The microarchitecture of a hair cell synapse may be such that the opening of a single Ca2+ channel evokes the simultaneous exocytosis of multiple synaptic vesicles. Thus, the differing demands of sensory encoding in the eye and ear generate diverse designs and capabilities for their ribbon synapses.

Short-Term Depression at the Reciprocal Synapses between a Retinal Bipolar Cell Terminal and Amacrine Cells

Visual adaptation is thought to occur partly at retinal synapses that are subject to plastic changes. However, the locus and properties of this plasticity are not well known. Here, we studied short-term plasticity at the reciprocal synapse between bipolar cell terminals and amacrine cells in goldfish retinal slices. Depolarization of a single bipolar cell terminal for 100 ms triggers the release of glutamate onto amacrine cell processes, which in turn leads to GABAergic feedback from amacrine cells onto the same terminal. We find that this release of GABA undergoes paired-pulse depression (PPD) that recovers in <1 min (single exponential time constant, τ ≅ 12 s). This disynaptic PPD is independent of mGluR-mediated plasticity and depletion of glutamatergic synaptic vesicle pools, because exocytosis assayed via capacitance jumps (ΔCm) recovered completely after 10 s (τ ≅ 2 s). Fast application of GABA (10 mm) onto outside-out patches excised from bipolar cell terminals showed that the recovery of GABAA receptors from desensitization depends on the duration of the application [fast recovery (<2 s) for short applications; slow (τ ≅ 12 s) for prolonged applications]. We thus blocked GABAA receptors and retested the GABAergic response mediated by nondesensitizing GABAC receptors to two rapid glutamate puffs onto the bipolar cell terminal. These responses consistently displayed PPD. Furthermore, blocking AMPA-receptor desensitization with cyclothiazide, or evoking GABA release with NMDA receptors, did not reduce PPD. We thus suggest that depletion of synaptic vesicle pools in GABAergic amacrine cells is a major contributor to PPD.

Glycinergic input to carp retinal ganglion cells may be mediated by glycine receptors with homologous kinetics.

Current responses of carp retinal ganglion cells (RGCs) retrogradely labeled and freshly dissociated to rapid application of glycine were recorded by whole-cell patch clamp techniques and effects of glycine antagonists on these responses were analyzed. The current response to maintained application of glycine at a concentration higher than 30 microM exhibited desensitization, which was well fitted to a monoexponential function. Strychnine (1 microM), a glycine receptor antagonist, completely blocked the response to 100 microM glycine. Strychnine at a concentration range between 10 and 200 nM suppressed the response to 100 microM glycine in a dose-dependent manner, and only a slow-activated and sustained current eventually remained in the presence of 200 nM strychnine. Power spectral density (PSD) analysis revealed no changes in the density-frequency dependence caused by strychnine. It was further shown that dissociation of strychnine from glycine receptors was rather slow. Moreover, Zn(2+) exerted similar dual action on this sustained response and the response in Ringers: potentiating and reducing them at low and high concentrations of Zn(2+), respectively. 5,7-Dichlorokynurenic acid (DCKA, 500 microM), a selective blocker of the glycine recognition site at the NMDA receptor, partially reduced the glycine response, but without changing its kinetics. These results suggest that glycinergic input to carp ganglion cells may be mediated by strychnine-sensitive glycine receptors with homologous kinetics, and slow dissociation of strychnine from glycine receptors may partially account for the changes in glycine response kinetics occurring in the presence of strychnine.

Probing electrical tuning of hair cells with a Zap current method in the intact amphibian papilla of bullfrogs.

Most, if not all, modern vertebrate species have evolved exquisite inner ears to discriminate acoustic signals of different frequencies, through a process called frequency tuning. For non‐mammalian species, at least part of frequency tuning has been attributed to intrinsic electrical properties of hair cells, i.e. electrical tuning. Since it was first discovered, the traditional method to assess electrical tuning has been to inject step current into hair cells and examine dampened membrane voltage oscillation. However, this method is not applicable for hair cells that do not oscillate. In this study, we developed a Zap current method that can be unbiasedly applied to all hair cells regardless of their oscillating behavior. Similar to a chirp sound in acoustic stimulation, a Zap current is a sinusoidal current with the frequency increased linearly with time. We first validated this new method with the traditional step current method on hair cells with dampened membrane voltage oscillation, and then applied it to all hair cells in the intact amphibian papilla of bullfrogs. We found that while hair cells with dampened membrane voltage oscillation are sharply tuned, non‐oscillating hair cells are broadly tuned. In addition, we found a third type of hair cells, which oscillate continuously and are extremely sharply tuned, with multiple peaks that are reminiscent of harmonics in the mammalian cochlea. In conclusion, the new Zap current method provides an unbiased way to assess electrical tuning, and it reveals an underappreciated heterogeneity of electrical tuning in the bullfrog amphibian papilla.