Ion channels in presynaptic nerve terminals and control of transmitter release.

The primary function of the presynaptic nerve terminal is to release transmitter quanta and thus activate the postsynaptic target cell. In almost every step leading to the release of transmitter quanta, there is a substantial involvement of ion channels. In this review, the multitude of ion channels in the presynaptic terminal are surveyed. There are at least 12 different major categories of ion channels representing several tens of different ion channel types; the number of different ion channel molecules at presynaptic nerve terminals is many hundreds. We describe the different ion channel molecules at the surface membrane and inside the nerve terminal in the context of their possible role in the process of transmitter release. Frequently, a number of different ion channel molecules, with the same basic function, are present at the same nerve terminal. This is especially evident in the cases of calcium channels and potassium channels. This abundance of ion channels allows for a physiological and pharmacological fine tuning of the process of transmitter release and thus of synaptic transmission.     

Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse

Synaptic communication requires proper coupling between voltage-gated Ca(2+) (Ca(V)) channels and synaptic vesicles. In photoreceptors, L-type Ca(V) channels are clustered close to synaptic ribbon release sites. Although clustered, Ca(V) channels move continuously within a confined domain slightly larger than the base of the ribbon. We hypothesized that expanding Ca(V) channel confinement domains should increase the number of channel openings needed to trigger vesicle release. Using single-particle tracking techniques, we measured the expansion of Ca(V) channel confinement domains caused by depletion of membrane cholesterol with cholesterol oxidase or methyl-β-cyclodextrin. With paired whole cell recordings from cones and horizontal cells, we then determined the number of Ca(V) channel openings contributing to cone Ca(V) currents (I(Ca)) and the number of vesicle fusion events contributing to horizontal cell excitatory postsynaptic currents (EPSCs) following cholesterol depletion. Expansion of Ca(V) channel confinement domains reduced the peak efficiency of release, decreasing the number of vesicle fusion events accompanying opening of each Ca(V) channel. Cholesterol depletion also inhibited exocytotic capacitance increases evoked by brief depolarizing steps. Changes in efficiency were not due to changes in I(Ca) amplitude or glutamate receptor properties. Replenishing cholesterol restored Ca(V) channel domain size and release efficiency to control levels. These results indicate that cholesterol is important for organizing the cone active zone. Furthermore, the finding that cholesterol depletion impairs coupling between channel opening and vesicle release by allowing Ca(V) channels to move further from release sites shows that changes in presynaptic Ca(V) channel mobility can be a mechanism for adjusting synaptic strength.     

Presynaptic regulation of quantal size: K+/H+ exchange stimulates vesicular glutamate transport

The amount of neurotransmitter stored in a single synaptic vesicle can determine the size of the postsynaptic response, but the factors that regulate vesicle filling are poorly understood. A proton electrochemical gradient (Δμ(H+)) generated by the vacuolar H(+)-ATPase drives the accumulation of classical transmitters into synaptic vesicles. The chemical component of Δμ(H+) (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters as well as in protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. However, synaptic vesicle uptake of the principal excitatory transmitter glutamate depends on the electrical component of Δμ(H+) (Δψ). We found that rat brain synaptic vesicles express monovalent cation/H(+) exchange activity that converts ΔpH into Δψ, and that this promotes synaptic vesicle filling with glutamate. Manipulating presynaptic K(+) at a glutamatergic synapse influenced quantal size, indicating that synaptic vesicle K(+)/H(+) exchange regulates glutamate release and synaptic transmission.     

Presynaptic calcium influx,neurotransmitter release,and neuromuscular disease

The synapse between a spinal motor neuron and a muscle cell is normally very effective at eliciting muscle contraction. A reliable connection between these two cells occurs because a single action potential reaching the motor nerve terminal normally releases hundreds of packets of transmitter containing thousands of chemical transmitter molecules, which cross the synapse and encounter a specialized region of postsynaptic muscle. Within the muscle membrane are thousands of receptor proteins specific for this transmitter. Activation of these postsynaptic receptors allows positively charged ions to cross the muscle membrane, generating a muscle cell action potential that leads to muscle contraction. Because of its size, contraction of a muscle cell requires the activation of an exceptionally large number of neurotransmitter receptors. To understand the regulation of this reliable communication and to elucidate details of pathological conditions that lead to muscle weakness, we have studied the subcellular mechanisms that govern synaptic transmission at the neuromuscular junction (NMJ). This article will review recent electron microscopic, electrophysiological, and imaging data in a discussion of the function of the motor nerve terminal in both normal and diseased states. Taken together, the existing data lead us to hypothesize that a small fraction of available calcium channels open within the transmitter releasing regions of the NMJ and that each vesicle fusion event is triggered by calcium flux through a single channel opening.     

AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis

AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.     

Fixation effects on synaptic vesicle density in neuromuscular junctions of young and old mice

It was previously reported that in soleus neuromuscular junctions of old mice, synaptic vesicle density was decreased while transmitter release was increased (compared to results in young mice). In the present study, two hypotheses that might resolve this disparity were tested. The first was that the density of readily releasable vesicles close to the preterminal membrane, rather than those in the whole terminal, would correlate with the physiological results. This hypothesis was excluded because both vesicle density in the 200 nm region just within the presynaptic terminal membrane, and total vesicle density were similarly reduced in old soleus junctions. The second hypothesis was that more transmitter was released during fixation at old than at young neuromuscular junctions, leading to an age-related depletion of vesicles. This was tested by counting vesicles in muscles fixed after transmission block was attained in Krebs solution lacking calcium, and by direct recording of quantal release during conventional fixation. This second hypothesis was excluded: in neuromuscular junctions exposed to zero-calcium Krebs solution before fixation, the age-related reduction in vesicle density was still present, and intracellular recording revealed only a slight increase in quantal transmitter release during fixation. Therefore, as discussed, other mechanisms must be considered.     

Evidence for the vesicle hypothesis

1. The relationship of synaptic vesicles to the synaptic cleft was examined with the electron microscope at neuromuscular junctions in the rat diaphragm before and after bathing the preparation in a physiological salt solution for 2 hr.2. A population of vesicles was defined which appeared to ;touch' the axoplasmic membrane. These vesicles were found to be aggregated adjacent to axoplasmic densities which lay opposite the mouths of post-synaptic junctional folds.3. Soaking in the salt solution and modifications of this solution increased the proportion of folds opposed by presynaptic densities with associated vesicles.4. Soaking in solutions with 20 mM-KCl depleted both the specific vesicle population and the whole population of terminal vesicles. The effect was shown in paired experiments to be a specific effect of the 20 mM-KCl, and it was prevented by a concomitant increase of the bathing MgCl(2) concentration.5. Soaking in solutions with a raised osmotic pressure reduced the specific but not the general vesicle population.6. It is suggested that these observations support the vesicle hypothesis and that the specific vesicle population forms part of a feed-back mechanism adjusting transmitter synthesis and mobilization to the rate of release of transmitter.     

Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze-fracture technique.

1. Structural changes which normally accompany transmitter release at frog neuromuscular junctions are visualized with the freeze-fracture technique. The effects of brown widow spider venom and botulinum toxin were evaluated in terms of their ability to block or produce these structural changes. Changes produced by these neuropoisons were correlated with their known effects on neurotransmitter release. 3. Fusion of synaptic vesicles with the presynaptic plasmalemma, normally evoked by electrical stimulation, was abolished at neuromuscular junctions from frogs treated with botulinum toxin. 3. The concentration of large intramembranous particles in the presynaptic plasmalemma, an indication of the excess of synaptic vesicle fusion over recovery of synaptic vesicle membrane, was increased by treatment with brown widow spider venom, even in the presence of botulinum toxin. 4. When external calcium was present, sites of vesicle fusion induced by brown widow spider venom, as well as by electrical stimulation, were located mainly in the active zone. In the absence of external calcium, many plasmalemmal deformations, also though to be sites of vesicle fusion, were more evenly dispersed over the presynaptic surface of nerve terminals. 5. Botulinum toxin decreased the number of vesicle fusion sites in the active zone induced by spider venom in the presence of external calcium but had little effect on the number of fusion sites induced by spider venom in the absence of external calcium. 6. Nerve terminals soaked in a sodium-free Ringer solution were partially depleted of vesicles. Addition of spider venom to this Ringer did not cause additional depletion of vesicles. 7. Formation of cation-permeable channels in the presynaptic membrane could account for these effects of spider venom on the frog neuromuscular junction. Botulinum toxin blocks vesicle fusion by some means which is not yet understood.     

Release from the cone ribbon synapse under bright light conditions can be controlled by the opening of only a few Ca(2+) channels

Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca(2+) channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca(2+) channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca(2+) currents (I(Ca)) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca(2+) channel number and single-channel current amplitude were calculated by mean-variance analysis of I(Ca). Two different comparisons-one comparing average numbers of release events to average I(Ca) amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone I(Ca)-suggested that fewer than three Ca(2+) channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca(2+) channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca(2+) dependence of release, Ca(2+) channel number, and Ca(2+) channel properties. The model replicated observations when a barrier was added to slow Ca(2+) diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca(2+) buffers did not affect release efficiency. The tight clustering of Ca(2+) channels, along with a high-Ca(2+) affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca(2+) influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light.     

N type Ca2+ channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes

Fast neurotransmitter release at presynaptic terminals occurs at specialized transmitter release sites where docked secretory vesicles are triggered to fuse with the membrane by the influx of Ca²⁺ ions that enter through local N type (CaV2.2) calcium channels. Thus, neurosecretion involves two key processes: the docking of vesicles at the transmitter release site, a process that involves the scaffold protein RIM (Rab3A interacting molecule) and its binding partner Munc-13, and the subsequent gating of vesicle fusion by activation of the Ca²⁺ channels. It is not known, however, whether the vesicle fusion complex with its attached Ca²⁺ channels and the vesicle docking complex are parts of a single multifunctional entity. The Ca²⁺ channel itself and RIM were used as markers for these two elements to address this question. We carried out immunostaining at the giant calyx-type synapse of the chick ciliary ganglion to localize the proteins at a native, undisturbed presynaptic nerve terminal. Quantitative immunostaining (intensity correlation analysis/intensity correlation quotient method) was used to test the relationship between these two proteins at the nerve terminal transmitter release face. The staining intensities for CaV2.2 and RIM covary strongly, consistent with the expectation that they are both components of the transmitter release sites. We then used immunoprecipitation to test if these proteins are also parts of a common molecular complex. However, precipitation of CaV2.2 failed to capture either RIM or Munc-13, a RIM binding partner. These findings indicate that although the vesicle fusion and the vesicle docking mechanisms coexist at the transmitter release face they are not parts of a common stable complex.     

Dopamine neurons release transmitter via a flickering fusion pore

A key question in understanding mechanisms of neurotransmitter release is whether the fusion pore of a synaptic vesicle regulates the amount of transmitter released during exocytosis. We measured dopamine release from small synaptic vesicles of rat cultured ventral midbrain neurons using carbon fiber amperometry. Our data indicate that small synaptic vesicle fusion pores flicker either once or multiple times in rapid succession, with each flicker releasing approximately 25-30% of vesicular dopamine. The incidence of events with multiple flickers was reciprocally regulated by phorbol esters and staurosporine. Thus, dopamine neurons regulate the amount of neurotransmitter released by small synaptic vesicles by controlling the number of fusion pore flickers per exocytotic event. This mode of exocytosis is a potential mechanism whereby neurons can rapidly reuse vesicles without undergoing the comparatively slow process of recycling.     

Protein-protein interactions in neurotransmitter release

The arrival of a nerve impulse at a nerve terminal leads to the opening of voltage-gated Ca(2+) channels and a rapid influx of Ca(2+). The increase in Ca(2+) concentration at the active zone from the basal level of 100-200 mM triggers the fusion of docked synaptic vesicles, resulting in neurotransmitter release. A large number of proteins have been identified at nerve terminals and a cascade of protein-protein interactions has been suggested to be involved in the cycling of synaptic vesicle states. Functional studies in last half decade on synaptic-terminal proteins, including Ca(2+) channels, have revealed that the SNARE core complex, consisting of synaptobrevin VAMP, a synaptic vesicle-associated protein, syntaxin and SNAP-25, synaptic membrane-associated proteins, acts as the membrane fusion machinery and that proteins interacting with the SNARE complex play essential roles in synaptic vesicle exocytosis by regulating assembly and disassembly of the SNARE complex.     

pH-dependent fusion of synaptosomal membrane studied by fluorescence quenching method

We have found that both the synaptic vesicles (SV) and synaptic plasma membrane vesicles (SPM) have an activity to fuse with phosphatidylcoline/phosphatidylserine liposomes in a pH-dependent manner. The activity increases with decreases in extravesicular pH. At a pH lower than 4.0, the activity is almost steady at its maximum value, and there was a rapid drop around pH 5.5. The pH-dependent fusion was inhibited by proteolysis with trypsin; hence, at least in part, some membrane proteins play an important role in these pH-dependent fusion processes. To find specific markers, we screened various protein modifiers and found that anion channel blockers, stilbene derivatives (DIDS and SITS) and glibenclamide, affected the fusion process. DIDS and SITS decreased the fusion activity with an IC50 of 180 and 300 microM, respectively, whereas glibenclamide, on the contrary, increased it. From the results of an autoradiogram using 3H-tagged DIDS, a 30 kDa DIDS-binding protein was identified in the synaptic plasma membrane, which is possible to be responsible for the pH-dependent fusion.     

Short-term synaptic plasticity, simulation of nerve terminal dynamics, and the effects of protein kinase C activation in rat hippocampus

Phorbol esters are hypothesised to produce a protein kinase C (PKC)-dependent increase in the probability of transmitter release via two mechanisms: facilitation of vesicle fusion or increases in synaptic vesicle number and replenishment. We used a combination of electrophysiology and computer simulation to distinguish these possibilities. We constructed a stochastic model of the presynaptic contacts between a pair of hippocampal pyramidal cells that used biologically realistic processes and was constrained by electrophysiological data. The model reproduced faithfully several forms of short-term synaptic plasticity, including short-term synaptic depression (STD), and allowed us to manipulate several experimentally inaccessible processes. Simulation of an increase in the size of the readily releasable vesicle pool and the time of vesicle replenishment decreased STD, whereas simulation of a facilitation of vesicle fusion downstream of Ca²⁺ influx enhanced STD. Because activation of protein kinase C with phorbol ester enhanced STD of EPSCs in rat hippocampal slice cultures, we conclude that an increase in the sensitivity of the release process for Ca²⁺ underlies the potentiation of neurotransmitter release by PKC.     

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

Neurotransmitter depletion by bafilomycin is promoted by vesicle turnover

Accumulation of neurotransmitter into synaptic vesicles is powered by the vacuolar proton ATPase. We show here that, in brain slices, application of the H(+)-ATPase inhibitors bafilomycin or concanamycin does not efficiently deplete glutamatergic vesicles of transmitter unless vesicle turnover is increased. Simulations of vesicle energetics suggest either that bafilomycin and concanamycin act on the H(+)-ATPase from inside the vesicle, or that the vesicle membrane potential is maintained after the H(+)-ATPase is inhibited.     

Elementary properties of spontaneous fusion of peptidergic vesicles: fusion pore gating

The release of hormones and neurotransmitters by regulated exocytosis requires the delivery of secretory vesicles to the plasma membrane, where they dock and become primed for fusion with the plasma membrane. Upon stimulation a fusion pore is formed through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. After the cargo release the fusion pore either closes (kiss-and-run, transient exocytosis), fluctuates between an open and a closed state (for short times, fusion pore flickering, or for rather longer periods, 'pulsing pore') or expands irreversibly (full fusion exocytosis). In almost all secretory cells spontaneous secretion of vesicle cargo can be detected in the absence of stimulation. Spontaneous and stimulated exocytosis were thought to exhibit similar properties at elementary level, differing only in the probability of occurrence. However, recent studies indicate that spontaneous exocytosis differs from the stimulated one in many respects, therefore opening questions about the physiological role of spontaneous exocytosis. In this report we address the elementary properties of spontaneous and stimulated peptidergic vesicle discharge which appears to be modulated by fusion pore conductance (diameter) and fusion pore gating.     

Transmitter metabolism as a mechanism of synaptic plasticity: a modeling study

The nervous system adapts to experience by changes in synaptic strength. The mechanisms of synaptic plasticity include changes in the probability of transmitter release and in postsynaptic responsiveness. Experimental and neuropharmacological evidence points toward a third variable in synaptic efficacy: changes in presynaptic transmitter concentration. Several groups, including our own, have reported changes in the amplitude and frequency of postsynaptic (miniature) events indicating that alterations in transmitter content cause alterations in vesicular transmitter content and vesicle dynamics. It is, however, not a priori clear how transmitter metabolism will affect vesicular transmitter content and how this in turn will affect pre- and postsynaptic functions. We therefore have constructed a model of the presynaptic terminal incorporating vesicular transmitter loading and the presynaptic vesicle cycle. We hypothesize that the experimentally observed synaptic plasticity after changes in transmitter metabolism puts predictable restrictions on vesicle loading, cytoplasmic-vesicular transmitter concentration gradient, and on vesicular cycling or release. The results of our model depend on the specific mechanism linking presynaptic transmitter concentration to vesicular dynamics, that is, alteration of vesicle maturation or alteration of release. It also makes a difference whether differentially filled vesicles are detected and differentially processed within the terminal or whether vesicle filling acts back onto the terminal by presynaptic autoreceptors. Therefore, the model allows one to decide, at a given synapse, how transmitter metabolism is linked to presynaptic function and efficacy.     

A new mechanism for antiepileptic drug action: vesicular entry may mediate the effects of levetiracetam

Levetiracetam (LEV) is one of the most commonly prescribed antiepileptic drugs, but its mechanism of action is uncertain. Based on prior information that LEV binds to the vesicular protein synaptic vesicle protein 2A and reduces presynaptic neurotransmitter release, we wanted to more rigorously characterize its effect on transmitter release and explain the requirement for a prolonged incubation period for its full effect to manifest. During whole cell patch recordings from rat hippocampal pyramidal neurons in vitro, we found that LEV decreased synaptic currents in a frequency-dependent manner and reduced the readily releasable pool of vesicles. When we manipulated spontaneous activity and stimulation paradigms, we found that synaptic activity during LEV incubation alters the time at which LEV's effect appears, as well as its magnitude. We believe that synaptic activity and concomitant vesicular release allow LEV to enter recycling vesicles to reach its binding site, synaptic vesicle protein 2A. In support of this hypothesis, a vesicular "load-unload" protocol using hypertonic sucrose in the presence of LEV quickly induced LEV's effect. The effect rapidly disappeared after unloading in the absence of LEV. These findings are compatible with LEV acting at an intravesicular binding site to modulate the release of transmitter and with its most marked effect on rapidly discharging neurons. Our results identify a unique neurobiological explanation for LEV's highly selective antiepileptic effect and suggest that synaptic vesicle proteins might be appropriate targets for the development of other neuroactive drugs.     

Diurnal changes in exocytosis and the number of synaptic ribbons at active zones of an ON-type bipolar cell terminal

The number and morphology of synaptic ribbons at photoreceptor and bipolar cell terminals has been reported to change on a circadian cycle. Here we sought to determine whether this phenomenon exists at goldfish Mb-type bipolar cell terminals with the aim of exploring the role of ribbons in transmitter release. We examined the physiology and ultrastructure of this terminal around two time points: midday and midnight. Nystatin perforated-patch recordings of membrane capacitance (C(m)) revealed that synaptic vesicle exocytosis evoked by short depolarizations was reduced at night, even though Ca(2+) currents were larger. The efficiency of exocytosis (measured as the DeltaC(m) jump per total Ca(2+) charge influx) was thus significantly lower at night. The paired-pulse ratio remained unchanged, however, suggesting that release probability was not altered. Hence the decreased exocytosis likely reflects a smaller readily releasable vesicle pool at night. Electron microscopy of single sections from intact retinas averaged 65% fewer ribbons at night. Interestingly, the number of active zones did not change from day to night, only the probability of finding a ribbon at an active zone. Additionally, synaptic vesicle halos surrounding the ribbons were more completely filled at night when these on-type bipolar cells are more hyperpolarized. There was no change, however, in the physical dimensions of synaptic ribbons from day to night. These results suggest that the size of the readily releasable vesicle pool and the efficiency of exocytosis are reduced at night when fewer ribbons are present at bipolar cell terminal active zones.