Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Endocytosis at ribbon synapses

Unlike conventional synaptic terminals that release neurotransmitter episodically in response to action potentials, neurons of the visual, auditory and vestibular systems encode sensory information in graded signals that are transmitted at their synapses by modulating the rate of continuous release. The synaptic ribbon, a specialized structure found at the active zones of these neurons, is necessary to sustain the high rates of exocytosis required for continuous release. To maintain the fidelity of synaptic transmission, exocytosis must be balanced by high-capacity endocytosis, to retrieve excess membrane inserted during vesicle fusion. Capacitance measurements following vesicle release in ribbon-type neurons indicate two kinetically distinct phases of compensatory endocytosis, whose relative contributions vary with stimulus intensity. The two phases can be independently regulated and may reflect different underlying mechanisms operating on separate pools of recycling vesicles. Electron microscopy shows diversity among ribbon-type synapses in the relative importance of clathrin-mediated endocytosis versus bulk membrane retrieval as mechanisms of compensatory endocytosis. Ribbon synapses, like conventional synapses, make use of multiple endocytosis pathways to replenish synaptic vesicle pools, depending on the physiological needs of the particular cell type.     

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca²⁺) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.     

Kinetics of Synaptic Transmission at Ribbon Synapses of Rods and Cones

The ribbon synapse is a specialized structure that allows photoreceptors to sustain the continuous release of vesicles for hours upon hours and years upon years but also respond rapidly to momentary changes in illumination. Light responses of cones are faster than those of rods and, mirroring this difference, synaptic transmission from cones is also faster than transmission from rods. This review evaluates the various factors that regulate synaptic kinetics and contribute to kinetic differences between rod and cone synapses. Presynaptically, the release of glutamate-laden synaptic vesicles is regulated by properties of the synaptic proteins involved in exocytosis, influx of calcium through calcium channels, calcium release from intracellular stores, diffusion of calcium to the release site, calcium buffering, and extrusion of calcium from the cytoplasm. The rate of vesicle replenishment also limits the ability of the synapse to follow changes in release. Post-synaptic factors include properties of glutamate receptors, dynamics of glutamate diffusion through the cleft, and glutamate uptake by glutamate transporters. Thus, multiple synaptic mechanisms help to shape the responses of second-order horizontal and bipolar cells.     

Protein composition of immunoprecipitated synaptic ribbons

The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis.     

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

Cholesterol as a key player in the balance of evoked and spontaneous glutamate release in rat brain cortical synaptosomes

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-β-cyclodextrin (MβCD) induced glutamate release in a dose-dependent fashion. HγCD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of MβCD on glutamate release induced by depolarizing stimuli, we observed that MβCD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by MβCD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with MβCD in the absence of calcium. We therefore investigated whether the cholesterol removal by MβCD changes intrasynaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if MβCD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by MβCD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by MβCD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals.     

Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B

First proposed as a specialized mode of release at sensory neurons possessing ribbon synapses, multivesicular release has since been described throughout the central nervous system. Many aspects of multivesicular release remain poorly understood. We explored mechanisms underlying simultaneous multivesicular release at ribbon synapses in salamander retinal rod photoreceptors. We assessed spontaneous release presynaptically by recording glutamate transporter anion currents (I_A(glu)) in rods. Spontaneous I_A(glu) events were correlated in amplitude and kinetics with simultaneously measured miniature excitatory postsynaptic currents in horizontal cells. Both measures indicated that a significant fraction of events is multiquantal, with an analysis of I_A(glu) revealing that multivesicular release constitutes ∼30% of spontaneous release events. I_A(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small I_A(glu) events were identical as were rise times of large and small miniature excitatory postsynaptic currents, indicating that the release of multiple vesicles during large events is highly synchronized. Effects of exogenous Ca²⁺ buffers suggested that multiquantal, but not uniquantal, release occurs preferentially near Ca²⁺ channels clustered beneath synaptic ribbons. Photoinactivation of ribbons reduced the frequency of spontaneous multiquantal events without affecting uniquantal release frequency, showing that spontaneous multiquantal release requires functional ribbons. Although both occur at ribbon-style active zones, the absence of cross-depletion indicates that evoked and spontaneous multiquantal release from ribbons involve different vesicle pools. Introducing an inhibitory peptide into rods to interfere with the SNARE protein, syntaxin 3B, selectively reduced multiquantal event frequency. These results support the hypothesis that simultaneous multiquantal release from rods arises from homotypic fusion among neighboring vesicles on ribbons and involves syntaxin 3B.     

Ca(2+) influx and neurotransmitter release at ribbon synapses

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.     

Hair cell ribbon synapses

Hearing and balance rely on the faithful synaptic coding of mechanical input by the auditory and vestibular hair cells of the inner ear. Mechanical deflection of their stereocilia causes the opening of mechanosensitive channels, resulting in hair cell depolarization, which controls the release of glutamate at ribbon-type synapses. Hair cells have a compact shape with strong polarity. Mechanoelectrical transduction and active membrane turnover associated with stereociliar renewal dominate the apical compartment. Transmitter release occurs at several active zones along the basolateral membrane. The astonishing capability of the hair cell ribbon synapse for temporally precise and reliable sensory coding has been the subject of intense investigation over the past few years. This research has been facilitated by the excellent experimental accessibility of the hair cell. For the same reason, the hair cell serves as an important model for studying presynaptic Ca(2+) signaling and stimulus-secretion coupling. In addition to common principles, hair cell synapses differ in their anatomical and functional properties among species, among the auditory and vestibular organs, and among hair cell positions within the organ. Here, we briefly review synaptic morphology and connectivity and then focus on stimulus-secretion coupling at hair cell synapses.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Synapsins in the vertebrate retina: absence from ribbon synapses and heterogeneous distribution among conventional synapses

The vertebrate retina contains two ultrastructurally distinct types of vesicle-containing synapses: conventional synapses, made predominantly by amacrine cells, and ribbon synapses, formed by photoreceptor and bipolar cells. To identify molecular differences between these synapse types, we have compared the distribution of the synapsins, a family of nerve terminal phosphoproteins, with that of synaptophysin (p38) and SV2, two intrinsic membrane proteins of synaptic vesicles. We report an absence of synapsin I and II immunoreactivity from all ribbon-containing nerve terminals. These include terminals of rod cells in developing and adult rat retina, rod and cone cells in monkey and salamander retinas, and rat bipolar cells. Furthermore, we show that synapsins I and II are differentially distributed among conventional synapses of amacrine cells. The absence of the synapsins from ribbon synapses suggests that vesicle clustering and mobilization in these terminals differ from that in conventional synapses.     

Ribbon Synapse Plasticity in the Cochleae of Guinea Pigs after Noise-Induced Silent Damage

Noise exposure at low levels or low doses can damage hair cell afferent ribbon synapses without causing permanent threshold shifts. In contrast to reports in the mouse cochleae, initial damage to ribbon synapses in the cochleae of guinea pigs is largely repairable. In the present study, we further investigated the repair process in ribbon synapses in guinea pigs after similar noise exposure. In the control samples, a small portion of afferent synapses lacked synaptic ribbons, suggesting the co-existence of conventional no-ribbon and ribbon synapses. The loss and recovery of hair cell ribbons and post-synaptic densities (PSDs) occurred in parallel, but the recovery was not complete, resulting in a permanent loss of less than 10% synapses. During the repair process, ribbons were temporally separated from the PSDs. A plastic interaction between ribbons and postsynaptic terminals may be involved in the reestablishment of synaptic contact between ribbons and PSDs, as shown by location changes in both structures. Synapse repair was associated with a breakdown in temporal processing, as reflected by poorer responses in the compound action potential (CAP) of auditory nerves to time-stress signals. Thus, deterioration in temporal processing originated from the cochlea. This deterioration developed with the recovery in hearing threshold and ribbon synapse counts, suggesting that the repaired synapses had deficits in temporal processing.     

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.     

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.     

Molecular anatomy and physiology of exocytosis in sensory hair cells

Hair cells mediate our senses of hearing and balance by synaptic release of glutamate from somatic active zones (AZs). They share conserved mechanisms of exocytosis with neurons and other secretory cells of diverse form and function. Concurrently, AZs of these neuro-epithelial hair cells employ several processes that differ remarkably from those of neuronal synaptic terminals of the brain. Their unique molecular anatomy enables them to better respond to small, graded changes in membrane potential and to produce unsurpassed rates of exocytosis. Here, we focus on the AZs of cochlear inner hair cells (IHCs). As in other hair cells, these AZs are occupied by a cytoplasmic extension of the presynaptic density, called the synaptic ribbon: a specialized protein complex required for normal physiological function. Some proteins found at IHC synapses are uniquely expressed or enriched there, where their disruption can beget deafness in humans and in animal models. Other proteins, essential for regulation of conventional neuronal Ca(2+)-triggered fusion, are apparently absent from IHCs. Certain common synaptic proteins appear to have extra significance at ribbon-type AZs because of their interactions with unique molecules, their unusual concentrations, or their atypical localization and regulation. We summarize the molecular-anatomical specializations that underlie the unique synaptic physiology of hair cells.     

Molecular organization and plasticity of the cytomatrix at the active zone

Regulated neurotransmitter release from presynaptic boutons is crucial for the functioning of chemical synapses, what in turn governs the functional performance of the nervous system. Release occurs at the active zone (AZ), a specialized region of the presynaptic plasma membrane that is defined by a unique and complex meshwork of proteins--the cytomatrix at the AZ (CAZ). Important functions of CAZ proteins include recruitment, docking and priming of synaptic vesicles as well as appropriate localization of voltage-gated calcium channels near vesicle docking sites. We will discuss recent progress in the understanding of the topological localization and the molecular functions of characteristic CAZ proteins as well as emerging molecular mechanisms underlying presynaptic plasticity that involve significant structural CAZ remodeling.     

The diverse roles of ribbon synapses in sensory neurotransmission

Sensory synapses of the visual and auditory systems must faithfully encode a wide dynamic range of graded signals, and must be capable of sustained transmitter release over long periods of time. Functionally and morphologically, these sensory synapses are unique: their active zones are specialized in several ways for sustained, rapid vesicle exocytosis, but their most striking feature is an organelle called the synaptic ribbon, which is a proteinaceous structure that extends into the cytoplasm at the active zone and tethers a large pool of releasable vesicles. But precisely how does the ribbon function to support tonic release at these synapses? Recent genetic and biophysical advances have begun to open the 'black box' of the synaptic ribbon with some surprising findings and promise to resolve its function in vision and hearing.     

Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca2+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+ channels and vesicles, and (2) promote vesicle replenishment.     

The expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina

In the present study, we generated a systematic overview of the expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina. Using indirect immunofluorescence microscopy, we analyzed the spatial and temporal distribution of 11 important membrane and membrane-associated synaptic proteins (syntaxin 1/3, SNAP-25, synaptobrevin 2, synaptogyrin, synaptotagmin I, SV2A, SV2B, Rab3A, clathrin light chains, CSP and neuroligin I) during synaptogenesis. The temporospatial distribution of these synaptic proteins was "normalized" by the simultaneous visualization of the synaptic vesicle protein synaptophysin, which served as an internal reference protein. We found that expression of various synaptic membrane proteins started at different time points and changed progressively during development. At early stages of development synaptic vesicle membrane proteins at extrasynaptic locations did not always colocalize with synaptophysin, indicating that these proteins probably do not reside in the same transport vesicles. Despite a non-synchronized onset of protein expression, clustering and colocalization of all synaptic membrane proteins at ribbon synapses roughly occurred in the same time window (between day 4 after birth, P4, and P5). Thus, the basic synaptic membrane machinery is already present in ribbon synapses before the well-known complete morphological maturation of ribbon synapses between P7 and P12. We conclude that ribbon synapse formation is a multistep process in which the concerted recruitment of synaptic membrane proteins is a relatively early event and clearly not the final step.